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Numerous studies have demonstrated the role of regulatory T (Treg) cells in peripheral tolerance. Nevertheless, how the survival and death of Treg cells is controlled is largely unknown. In this study, we investigated the mechanisms involved in regulating the homeostasis of a subset of Ag-specific αβTCR+ CD4−CD8− double negative (DN) Treg cells. We demonstrate that DN Treg cells are naturally resistant to TCR cross-linking-induced apoptosis. Administration of exogenous IL-10 renders DN Treg cells susceptible to apoptosis, and abolishes their suppressive function. Furthermore, TCR cross-linking of DN Treg cells in the presence of IL-10 leads to the up-regulation of the membrane-bound but not the soluble form of TNF-α. Interaction of membrane bound TNF-α with TNFR2 sends death signals to DN Treg cells. Blocking their interaction can reverse the effects of IL-10 on DN Treg cells. These results provide insights into the mechanisms that regulate the function and homeostasis of DN Treg cells.

The regulation of apoptosis in mature CD4+ or CD8+ αβ+ T cells has been well studied. How the survival and death is regulated in peripheral CD4−CD8− (double negative, DN) αβ+ T cells remains unknown. Recent studies suggest that peripheral DN T cells may play an important role in the regulation of the immune responses mediated by CD4+ or CD8+ T cells. Here, we used immunosuppressive DN T cell clones to elucidate the mechanisms involved in the regulation of death and survival of αβ+ DN T cells. The DN T cell clones were generated from the spleen cells of 2C transgenic mice, which express the transgenic TCR specific for Ld and permanently accepted Ld+ skin allografts after pretransplant infusion of Ld+ lymphocytes. We report that 1) the mature DN T cells are highly resistant to TCR cross-linking-induced apoptosis in the presence of exogenous IL-4; 2) Fas/Fas-ligand and TNF-α/TNFR pathways do not play an apparent role in regulating apoptosis in DN T cells; 3) the DN T cells constitutively express a high level of Bcl-xL, but not Bcl-2; 4) both Bcl-xL and Bcl-2 are up-regulated following TCR-cross-linking; and 5) IL-4 stimulation significantly up-regulates Bcl-xL and c-Jun expression and leads to mitogen-activated protein kinase phosphorylation in DN T cells, which may contribute to the resistance to apoptosis in these T cells. Taken together, these results provide us with an insight into how mature DN T cells resist activation-induced apoptosis to provide a long-term suppressor function in vivo.

The distribution of stanniocalcin immunoreactivity was examined in the corpuscles of Stannius of the white sucker (Catostomus commersoni) by using a chum salmon stanniocalcin antiserum, Western blotting, and light and electron microscopy. The white sucker possesses at least two stanniocalcin-immunoreactive corpuscles in the most posterior portions of the kidneys. Immunocytochemistry and ultrastructure revealed two cell-types in the corpuscle parenchyma, only one of which was immunoreactive. The nonimmunoreactive cells contained dense-cored vesicles and long processes that extended between the immunoreactive cells and terminated at perivascular spaces. When corpuscle extracts were subjected to electrophoresis and Western blotting, three nonreduced stanniocalcin-like immunoreactive bands (approximately 56, 61, and 64 kDa) were observed. However, in the presence of a reductant, a diffuse band migrating in the range of 28 to 32 kDa was noted. The results of this study on the white sucker demonstrate the presence of a dimeric stanniocalcin-like molecule and present evidence of a previously uncharacterized cell-type in the corpuscles of Stannius.

A cDNA that codes for the polypeptide hormone precursor proopiomelanocortin (POMC) was cloned and sequenced from a gar (Lepisosteus osseus) pituitary cDNA library. The gar POMC cDNA is 1237 bp and contains a 780-bp open reading frame. The deduced amino acid sequence for gar POMC is 259 amino acids in length. The general organization of gar POMC is very similar to that of other gnathostome POMC sequences. The beta-endorphin sequence had 91% sequence identity with sockeye A beta-endorphin and 71% sequence identity with Xenopus laevis beta-endorphin. Three melanocyte-stimulating hormone (MSH) core sequences [HFR(W)] were detected. The gar alpha-MSH sequence was identical to the alpha-MSH sequence in rat POMC. The gar beta-MSH sequence had 77% sequence identity with salmonid forms of beta-MSH and 53% sequence identity with tetrapod forms of beta-MSH. The gamma-MSH region of gar POMC only had 26% primary sequence identity with tetrapod gamma-MSH sequences. Gar gamma-MSH had an incomplete MSH core sequence (HRF), an apparent internal deletion of five amino acids, and lacked flanking paired basic amino acids essential for proteolytic cleavage. The apparent degenerate nature of gar gamma-MSH is discussed in light of the absence of this sequence in salmonid fish.

An antiserum to cod somatolactin (SL) was used for immunohistochemical screening for the pars intermedia of two teleosts (Oreochromis mossambicus and Gymothorax meleagris), two holostean fishes (Lepisosteus osseus and Amia calva), and a chondrostean fish (Acipenser fulvescens) for SL-immunopositive (SL-IR) cells. As expected, a subset of the epithelial cells in the pars intermedia of O. mossambicus (tilapia) was immunopositive for SL, and the remainder of the epithelial cells was immunopositive for alpha-MSH-specific antiserum (alpha-MSH-IR). SL-IR was not detected in any of the epithelial cells in the pars intermedia of the moray eel G. meleagris. To determine whether SL-IR could be detected in nonteleost fishes, immunohistochemical analyses were done on the pituitaries of two holostean fishes and one chondrostean fish. In the pars intermedia of the gar, L. osseus, a subset of cells was immunopositive for alpha-MSH only. However, in the pars intermedia of the bowfin, A. calva, all of the epithelial cells indicated the presence of both SL and alpha-MSH. Finally, no SL-positive cells were detected in the pars intermedia of the sturgeon, A. fulvescens.

The holostean fish occupy an important position in vertebrate phylogeny as extant representatives of a ancient group of ray-finned fish with evolutionary connections to present-day teleosts. Incubation of heat-denatured plasma from the bowfin Amia calva with trypsin generated bradykinin-like immunoreactivity. The primary structure of bowfin bradykinin was established as Ala-Pro-Pro-Gly-Trp-Ser-Pro-Phe-Arg. This amino acid sequence contains one amino acid substitution (Phe 5 → Trp) compared with mammalian bradykinin. The same peptide was generated in heat-denatured plasma from the longnosed gar Lepisosteus osseus . Treatment of plasma from either the bowfin or gar with glass beads under conditions previously shown to activate Factor XII in the plasma of mammals and reptiles did not generate bradykinin. Bolus injections of synthetic bowfin bradykinin (0.1, 0.3, and 1.0 nmol/kg) into the bulbus arteriosus of unanesthetized bowfin resulted in an immediate fall in arterial blood pressure of 5–10 min duration that was followed by a dose-dependent rise in pressure that was sustained for 30–60 min. There was no change in heart rate following bradykinin administration. The data suggest that the kallikrein-kinin system may predate the appearance of teleosts and may play a role in cardiovascular regulation in holosteans.

Stanniocalcin-immunoreactive cells were localized in the corpuscles of Stannius of a holostean fish, the garpike (Lepisosteus osseus), using antisera against salmon and trout stanniocalcins and the peroxidase-antiperoxidase and protein A-gold immunohistochemical methods. The stanniocalcin-immunoreactive cells were periodic acid-Schiff-positive, and antibody staining was abolished if the antiserum was preabsorbed with corpuscle homogenate. Immunocytochemistry revealed two reactive cell types in the glandular parenchyma, and immunoreactivity was confined to the secretory granules. Staining of the granules was also abolished when the antisera were blocked with crude corpuscle homogenate. When corpuscle extracts from garpike were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis, a single dense band was evident with a molecular weight of ∼68 kDa under non-reducing conditions, whereas three bands were observed (∼29, ∼31, and ∼34 kDa) under reducing conditions. Staining of all bands disappeared following preabsorption of the antiserum with salmon stanniocalcin, trout stanniocalcin, or garpike corpuscle extract. The results are compared with stanniocalcins from another extant holostean, the bowfin (Amia calva), and from more modern bony fishes, the teleosts.

Acid extracts of the intermediate pituitaries of the gars, L. spatula and L. osseus , were fractionated by Sephadex G-50 column chromatography and analyzed by radioimmunoassay. This procedure revealed that immunoreactive forms of N- acetylated β-endorphin- and α-MSH-sized material were present in equimolar amounts and represented the major end products of the POMC biosynthetic pathway in these species. Cation-exchange chromatography indicated that multiple N- acetylated forms of β-endorphin were present in the intermediate pituitaries of the two species of gar, and that these forms differed in their net positive charge and in their apparent molecular weight. Reversed-phase HPLC analysis of the α-MSH-related material indicated that up to 90% of the total MSH in the pituitary of the gar was N- acetylated . Furthermore, the predominant form of α-MSH in both species of gar was N,O- diacetyl-ACTH (1–13)- NH 2 . Nearly identical results were obtained following the analysis of α-MSH-related peptides in the intermediate pituitary of the bowfin, A. calva . The pattern of posttranslational processing of POMC observed in the intermediate pituitaries of holostean fishes is very similar to the processing events observed in lungfishes, turtles, and mammals; hence, the processing of POMC has been remarkably conserved during vertebrate evolution.

Stanniocalcin (STC) is a homodimeric glycoprotein hormone implicated in calcium and phosphate regulation in both teleost fish and mammals. In the present study, immunostaining with salmon STC antiserum demonstrated that STC cells were localized in both the corpuscles of Stannius (CS) and in specific cells of the distal renal tubules of the silver arawana, Osteoglossum bicirrhosum , an ancient ray-finned fish (actinoptergian) and basal teleost (Order Osteoglossiforme). The morphology of these STC-immunoreactive kidney cells was similar to renal ‘chloride’ (mitochondrial-rich) cells. The immunoreactive renal cells were present in two of three other osteoglossiformes and absent in the gar, a nonteleost actinopterygian and the eel, another basal teleost. The arawana STC cDNA encodes a prehormone of 249 amino acids (aa) with a signal peptide of 31 aa and a mature protein of 218 aa. The deduced aa sequence of arawana STC shows 54–66% identity with other teleost STCs and 51–52% identity with mammalian STC-1. The deduced aa sequence of arawana STC contains ten cysteines, compared with 11 in teleost STC and in mammalian STC-1. The cysteine substitution occurs at the site of inter-monomeric disulfide linkage. Western blot analysis revealed a single 21 kDa band under non-reducing conditions, and a single band of 25 kDa under reducing conditions. These data indicate that arawana STC exists as a monomeric peptide. Northern blot analysis detected a 3.3 kb STC mRNA confined to the CS, with no hybridization signal in either the remaining kidney or in gut, muscle, brain and heart. The significance of the STC signal in cells of the renal tubules of arawana and two other Osteoglossiforme species requires further investigation. This is the first report of a monomeric form of STC in any vertebrate and the first evidence of STC in renal tubules of adult fish.