Identification and characterization of two members of a novel class of the interleukin-1 receptor (IL-1R) family. Delineation of a new class of IL-1R-related proteins based on signaling.

Two novel members of the interleukin-1 receptor (IL-1R) family, identified by homology searches of human genomic sequence data bases, are described. The genes have been named according to their structural features: TIGIRR-1 (three immunoglobulin domain-containing IL-1 receptor-related) and TIGIRR-2. TIGIRR-2 has recently been identified as causing mental retardation when mutated (Carrie, A., Jun, L., Bienvenu, T., Vinet, M. C., McDonell, N., Couvert, P., Zemni, R., Cardona, A., Van Buggenhout, G., Frints, S., Hamel, B., Moraine, C., Ropers, H. H., Strom, T., Howell, G. R., Whittaker, A., Ross, M. T., Kahn, A., Fryns, J. P., Beldjord, C., Marynen, P., and Chelly, J. (1999) Nat. Genet. 23, 25-31) and called IL1RAPL, a name we will also use henceforth. Neither receptor alone was able to mediate transcriptional activation of NF-kappaB in response to IL-1alpha, IL-1beta, or IL-18. In order to begin to elucidate the function of these and other orphan IL-1R family members, we have developed a functional assay utilizing a panel of chimeric receptors containing the extracellular and transmembrane domains of either type I IL-1R or IL-1R accessory protein (AcP) coupled to the cytoplasmic domains of all family members. Coexpression of each IL-1R chimera with each AcP chimera and an NF-kappaB-responsive reporter demonstrated that the cytoplasmic domains could be classified as IL-1R-like, AcP-like, or neither. Any IL-1R-like cytoplasmic domain could cooperate with any AcP-like cytoplasmic domain. The TIGIRR-1 and IL1RAPL cytoplasmic domains, however, were unable to signal as either IL-1R-like or AcP-like components, suggesting that they function as a new class of receptors within this family.

Functional characterization of the human interleukin-15 receptor alpha chain and close linkage of IL15RA and IL2RA genes.

Interleukins-2 and -15 (IL-2 and IL-15) are cytokines with overlapping but distinct biological effects. Their receptors share two subunits (the IL-2R beta and -gamma chains) that are essential for signal transduction. The IL-2 receptor requires an additional IL-2-specific alpha subunit for high affinity IL-2 binding. Recently, a murine IL-15-specific alpha subunit was identified, cloned, and shown to be structurally related to IL-2R alpha. However, the murine IL-15R alpha alone bound IL-15 with a 1000-fold higher affinity than that seen with IL-2R alpha and IL-2. We now extend these studies into the human system with the isolation of three differentially spliced human IL-15R alpha variants that are all capable of high affinity binding of IL-15. The cytoplasmic domain of IL-15R alpha, like that of IL-2R alpha, is dispensable for mitogenic signaling, suggesting that the primary role of the alpha chains is to confer high affinity binding. At high concentrations, IL-15, like IL-2, is able to signal through a complex of IL-2R beta and -gamma in the absence of the alpha subunit. Furthermore, the IL15RA and IL2RA genes have a similar intron-exon organization and are closely linked in both human and murine genomes. However, the distribution of expression of the IL-15R alpha is much wider than that of the IL-2R alpha, suggesting a broader range of cellular targets for IL-15.

Translational control of globin chain ontogeny in hamster yolk sac erythroid cells.

Prior research has demonstrated that globin ontogeny of hamster proceeds nearly to completion during the several days that yolk sac erythroid cells (YSEC) circulate in the embryo; synthesis of embryonic globin chains gives way to synthesis of adult globin chains in these primitive cells. In the present study, we translated total cell RNA extracted from YSEC on days 9-13 of gestation in wheat germ cell-free extract, expecting to observe the same progressive rise that occurs in vivo in rates of translation of alpha- and beta-globin mRNA during ontogeny. The opposite occurred; translation rates of both globins decreased sharply. This disparity between synthesis of alpha- and beta-globins in vivo and in vitro suggested an element of control of translation attributable to the YSEC cytoplasm. We therefore assayed the effect of RNA-free clarified YSEC cytoplasm on cell-free translation of YSEC RNA. A repression of translation was detected of alpha- and beta-globin mRNA (not of embryonic globin mRNA), exercised strongly by cytoplasm from YSEC early in ontogeny (gestational day 9), and weakening as ontogeny progressed. The same effect was noted on alpha- and beta-globin mRNA of adult hamster and of rabbit. Heat treatment of cytoplasm abolished the greater part of the translation regulation, suggesting that the active agent is protein. Further characterization of this translational regulator included: (a) it binds to globin poly(A) mRNA but not to poly(A), (b) it was not detected in cell lysate of adult hamster brain, lung, or erythrocytes, and (c) it did not inhibit translation of adult hamster brain and liver RNA. We conclude that hamster globin ontogeny is substantially modulated by this translational regulation of alpha- and beta-globin expression.

Erythropoietin. Receptor characteristics during the ontogeny of hamster yolk sac erythroid cells.

Erythropoietin (Epo) binds specifically to receptors on the surface membrane of responsive erythroid cells. In search of ontogenic changes in Epo receptor behavior, we studied characteristics of specific binding to hamster yolk sac erythroid cells during hamster ontogeny. We detected receptors specific for Epo on these cells throughout the duration of their intravascular existence (hamster gestational days 8 through 13). These receptors are saturable at an Epo concentration of 1.2 nM in the incubation medium. Attainment of equilibrium of binding prior to hormone internalization, a requirement for receptor binding assays, was possible at 10 degrees C but not at 37 degrees C. Hence, all incubations of cells with Epo were carried out at 10 degrees C. Data on specific binding analyzed by the method of Scatchard demonstrated that yolk sac erythroid cells possess a single class of Epo receptors at each stage of gestation examined. Binding affinity and numbers of receptors per cell change as ontogeny progresses: Kd (the dissociation constant) increases, a phenomenon observed in other differentiating cell systems, whereas the number of receptors per cell peaks on gestational day 10. The variability in number of receptors per cell is consonant with up and down regulation controlled by Epo availability. We propose that the progressive increase in Kd might be best explained by ontogenic changes in cell membrane structure contiguous to the receptors themselves.

The globin gene expression program in the hamster embryo.

Molecular mechanisms involved in control of globin gene expression are a prominent target in current basic biologic research. A better understanding of these mechanisms might also impinge on a clinical goal: amelioration of the human hemoglobinopathies. Recent reports have established the coexistence of embryonic and adult globins in rodent yolk-sac erythroid cells, raising the possibility that globin ontogeny takes place in these cells. The present study was undertaken to define the extent of this putative ontogenic process. We measured daily rates of synthesis of individual globins in hamster yolk-sac erythroid cells from the earliest day in gestation that these cells are available (day 7) until the day they cease to replicate (day 13). Converted to a per-cell basis, the rates demonstrate an ontogenic progression in globin synthesis, from embryonic globins to adult globins, that encompasses nearly entirely the total globin ontogeny of this mammal. Synthesis of adult alpha globin is already detectable on day 7, whereas synthesis of the two adult beta globins does not appear until day 9. Synthesis of embryonic y globin stands in contrast to that of the other two embryonic globins (x and z), rising as they fall, a phenomenon reminiscent of the gamma globin of primates and certain ruminants. This physiologic primitive erythroid cell appears to be an unusually appropriate target for studies directed at globin ontogeny.

Ontogeny of hamster hemoglobins in yolk-sac erythroid cells in vivo and in culture.

During mammalian hemoglobin ontogeny, synthesis of the earliest globin chains (embryonic) is ultimately replaced by synthesis of globin chains (adult) characteristic of the fully formed organism. Elements of control of initiation, progression, and completion of globin-chain ontogeny are poorly understood. In search of a cell culture system in which ontogeny might be studied under closely controlled experimental conditions, we chose erythroid cells of the hamster embryo. First, the ontogeny of globin chains was defined in these yolk-sac-derived erythroid cells from day 10 through day 13 in gestation. Amounts of individual embryonic and adult globin chains were quantified, as were their rates of synthesis. Next, analogous studies were performed on yolk-sac erythroid cells from day 10 in gestation (prior to the appearance of fetal liver) grown in culture for 3 days, corresponding to days 10-13 in vivo. The ontogenic program in culture was virtually identical to that in vivo. Approximately 70% of active globin synthesis was embryonic at day 10 in gestation (day 0 of culture), declining to 30% by day 13 in gestation (day 3 of culture). Whereas only trace synthesis of the adult non-alpha chains (beta major and beta minor) were initially observed, their combined active synthesis achieved a level of approximately 30% 3 days later both in vivo and in culture. Cell hemoglobin content and cell morphology were similar in both systems. We conclude that an ontogenic program for globin-chain synthesis exists in these primitive erythroid cells, overriding possible influences of cell environment. Further, we suggest that these cells in culture provide a means of examining cell mechanisms associated with globin-gene ontogeny under controlled experimental conditions.

Studies on globin chain synthesis during hamster development.

Erythroid cells derived from the yolk-sac of the hamster embryos replicate in the embryonic circulation, actively synthesize hemoglobin, and are the dominant circulating erythroid cell through day 14 of gestation. Until day 11, at which time fetal liver rudiments appear, they are the only erythroid tissue present in the embryo. Because of their size (Fig. 1 and Table 1) they can be separated from erythrocytes produced by ontogenically later hemopoietic tissue (liver, spleen, bone marrow). The embryonic (three) and adult (three) globin chains of hamster hemoglobins separate cleanly by electrophoresis in polyacrylamide gel, (Fig. 2). This provides an advantage over mouse, whose x and z chains tend to co-migrate. During hamster embryonic development, yolk-sac erythroid cells first become available on day 7. Nine days later (birth), globin-chain ontogeny in the circulating blood is nearly complete (Fig. 3). When yolk-sac-derived erythroid cells are isolated at day 10 in gestation (prior to the appearance of fetal liver) and grown in culture for 3 days, changes in types of globin chains synthesized are identical to globin-chain ontogenic changes in vivo during gestational days 10 through 13 (Fig. 5). We conclude that the ontogenic "switch" of globin-gene expression in these erythroid cells is pre-programmed, and that the program is capable of full function despite a drastic change in cell environment from circulating blood to culture medium. The ability to study these primitive erythroid cells under the tightly controllable conditions of culture might facilitate acquisition of information on mechanisms of globin-gene ontogeny.

Characteristics of I and i antigen receptors on the membrane of erythrocytes in sickle cell anemia.

In this study we employed two approaches in attempts to explain the increased expression of I and i antigens on the membrane surface of SS erythrocytes: immunoelectron microscopy and measurements of antigen-antibody affinity. AA and SS erythrocytes were first reacted with anti-I or anti-i antisera, and then with ferritin-conjugated anti-human IgM. With both antibodies, proportions of cells labeled and ferritin-cluster density on labeled cells were greater for SS erythrocytes at a high level of significance. Studies on the temperature dependence of agglutination by anti-I provided the association constants of agglutination, which were uniformly greater for SS erythrocytes. I and i antigen determinants are carried principally on band 3, the major transmembrane protein of the erythrocyte membrane. Therefore, for SS erythrocytes, the increased I (and i) antigen density and the increased association constant of agglutination by anti-I very likely both reflect the same phenomenon: a relatively greater exposure of I (and i) antigen determinant sites at the membrane surface. Any deviation from normal at the exterior surface of the erythrocyte membrane deserves to be suspect for potential pathogenicity, because this is the cell's region of intimate contact with other elements of the circulation. In addition to enhancing susceptibility of SS erythrocytes to cold agglutinins, an altered exposure of band 3 at the membrane outer surface has the potential for accelerating recognition and removal of SS erythrocytes from the circulation by mononuclear phagocytes.

Simultaneous expression of globin genes for embryonic and adult hemoglobins during mammalian ontogeny.

The dominant hemoglobin of the adult hamster was detected in yolk-sac erythroid cells, and its identity was confirmed by peptide mapping and by analysis of relevant peptides. Both the presence and active synthesis of two embryonic hemoglobins presumed to exist only in yolk-sac erythroid cells were detected in neonatal liver and spleen. Thus the time span of expression of both embryonic and adult globin genes during mammalian ontogeny may be considerably broader than presently believed.

Role of membrane lipids in cold agglutination of human erythrocytes.

The membrane lipid fluidity of normal human erythrocytes was modified by enrichment and depletion in cholesterol, and the expression of I and SP1 antigens was assayed by quantitative hemagglutination from 4 degrees to 24 degrees C by use of a continuous flow system. Below 16 degrees--18 degrees C, cholesterol enrichment increased and cholesterol depletion decreased percent agglutination. As temperatures approached approximately 18 degrees--20 degrees C, differences in agglutination between modified and unmodified erythrocytes became insignificant despite marked differences in lipid fluidity at that temperature. Thus, fluidity changes alone cannot be responsible for the effect of membrane cholesterol on cold agglutination. In an additional study, the temperature dependence of a relative equilibrium association constant, estimated by probit analysis of percent agglutination at various antisera concentrations, was biphasic with a sharp break at 16 degrees C. Our studies are consistent with the hypothesis that I and Sp1 antigens preferentially partition into a lipid domain that forms during lateral phase separation of membrane lipid developing at low temperature. A resulting increase in antigen density would then become responsible for augmented agglutination by specific antibody.

Variability of intracellular pH within individual populations of SS and AA erythrocytes.

Individual populations of AA and SS erythrocytes were fractionated according to cell density by centrifugation, and the fractions analysed for intracellular pH (PHi), the mole ratio of 2,3-diphosphoglycerate to haemoglobin (DPG:Hb), and cell concentration of haemoglobin (MCHC). The pHi of SS erythrocytes was consistently lower than that of AA erythrocytes throughout the density range, and the lowest pHi of both cell types (AA and SS) was found in cells with the highest density. As the highest density AA and SS erythrocytes are characterized by the lowest DPG:Hb values, their relatively low pHi cannot be ascribed to intracellular organic phosphate. Instead we propose that a redistribution of hydrogen ions across the membrane of both AA and SS erythrocytes is the ultimate result of progressive alterations in these membranes in vivo.

Fetal characteristics of erythrocytes in sickle cell anemia: an immunofluorescence study of individual cells.

In a group of disease states that includes sickle cell anemia (SS disease), two fetal erythrocyte markers, Hb F and i antigen, persist into adulthood. Using the technique of single-cell immunofluorescence, we determined the expression of l-i antigens and the presence of Hb F within populations of erythrocytes. Subjects tested included normal adults, normal newborns, patients with SS disease, and individuals with sickle cell trait. We classified erythrocytes reacting to anti-i as i cells and those reacting to anti-l as l cells, a terminology analogous to that used to identify erythrocytes containing increased amounts of Hb F as F cells. The expression of l and i antigens within populations of both normal and SS erythrocytes was found to be heterogeneous. The proporations of both i cells and l cells in all SS patients studied exceeded those found in normal adults, and an overall stronger-than-normal reactivity of individual SS cells to the two antibodies was observed. Proportions of F cells showed no correlation with proportions of i cells; and with double fluroescence staining for both Hb F and i, a significant proportion of each total SS red cell population was found to carry only one or the other marker. These findings confirm and clarify on a cellular level our previous demonstration, by means of quantitative hemagglutination, that there is increased expression of both l and i by whole populations of SS erythrocytes. In addition, we provide here new information on the expression of l and i within populations of normal human erythrocytes.

Increased expression in erythrocytic Ii antigens in sickle cell disease and sickle cell trait.

The reactivity of human erythrocytes with anti-I and anti-i sera was estimated in 36 normal adults, 18 individuals with sickle cell anemia and 27 individuals with sickle cell trait, by quantitative hemagglutination with a sensitive autoanalyzer system. SS erythrocytes showed significantly increased I and i reactivity when compared to normal erythrocytes. AS erythrocytes also demonstrated some increase in Ii reactivity. No correlation was found between erythrocytic HbF content and i reactivity.