Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.     

Immune proteasomes in the development of the rat immune system

The dynamics of the expression of LMP7 and LMP2 proteasome subunits during embryonic and early postnatal development of rat spleen and liver was studied in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The distribution of LMP7 and LMP2 immune subunits in spleen and liver cells was also evaluated throughout development. The common tendency of both organs to increase the expression of both LMP7 and LMP2 subunits on the 21st postnatal day (P21) was found. However, the total proteasome level was shown to be constant. At certain developmental stages, the dynamics of immune subunits expression in the spleen and liver was different. While the gradual enhancement of both immune subunits was observed on P1, P18 and P21 in the spleen, the periods of gradual increase observed on E16 (the 16th embryonic day) and E18 gave way to a period of decrease in immune subunits on P5 in the liver. This level did not reliably change until P18 and increased on P21. The revealed changes were accompanied by an increase in chymotrypsin-like activity and a decrease in caspase-like activity in the spleen at P21 compared to the embryonic period. This indicates the increase in proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased compared to the embryonic period by P21. The dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Moreover, it was discovered that the increase in the expression of immune subunits during early spleen development is associated with the process of formation of white pulp by B- and T-lymphocytes enriched with immune subunits. In the liver, the increase in the level of immune subunits by P21 was also accompanied by an increase of their expression in hepatocytes. While the decrease of their level by P5 may be associated with the fact that the liver has lost its function as the primary lymphoid organ in the immune system by this time, as well as with the disappearance of B-lymphocytes enriched with immune proteasomes. In the spleen and the liver, MHC class I molecules were found during the periods of increased levels of proteasome immune subunits. On E21, the liver was enriched with neuronal nitric oxide synthase (nNOS); the level of nNOS decreased after birth and then increased by P18. This fact indicates the possibility of the induction of expression of the LMP7 and LMP2 immune subunits in hepatocytes via a signaling pathway involving nNOS. These results indicate that compared to the rat liver cells, splenic T cell immune response develops in rats starting around P19–P21. First, a T-area of white pulp is formed in the spleen during this period. Second, an increased level of immune proteasomes and MHC class I molecules in hepatocytes can ensure the formation of antigenic epitopes from foreign proteins and their delivery to the cell surface for subsequent presentation to cytotoxic T-lymphocytes.     

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.     

Immune proteasomes in the developing rat spleen

Changes in the structure of the rat spleen and the distribution of immune proteasomes in it during early postnatal development have been studied using double immunofluorescent staining of tissue sections with antibodies to the LMP7 immune proteasome subunit and to specific markers of T and B lymphocytes. It has been shown that the white pulp on postnatal day 5 is not yet colonized by lymphocytes and contains a smaller amount of immune proteasomes than the red pulp. At this stage, T and B lymphocytes concentrate mainly in the red pulp. On day 8, B lymphocytes occupy the marginal zone, while T lymphocytes aggregate into dense strands close to the white pulp. By day 18, T lymphocytes form periarteriolar sheaths in the white pulp, and the contents of immune proteasomes in the red and white pulp become equally high. An increase in the total content of immune proteasomes in the spleen on the third postnatal week was revealed in our previous study by Western blotting. In addition to T and B lymphocytes, immune proteasomes have also been revealed in other spleen cell types, probably in macrophages and reticular cells of the white pulp. Thus, the postnatal development of the spleen is associated with an increase in the contents of immune proteasomes in it.     

Proteasomes on thyroid tissue allotransplantation under induction of donor-specific tolerance in rats

Proteasomes in the liver of August rats (RT1^c) were investigated 30 days after allotransplantation of Wistar rat (RT1^u) thyroid tissue under renal capsule with/without induction of donor-specific tolerance by donor splenocyte intraportal administration. The levels of total proteasome pool, immune proteasomes containing subunits LMP2 and/or LMP7, and proteasome regulators 19S and 11S were defined. Intact and sham-operated August rats were used as control groups. The level of all immune proteasome forms and 11S regulator increased while the level of the total proteasome pool and 19S regulator decreased in the liver of experimental animals compared to the control groups, which indicated changes of liver functional state after transplantation. The 19S/11S ratio increased in the liver of nontolerant rats compared to tolerant animals. In the liver of tolerant rats with accepted grafts, the number of mononuclear cells expressing the immune subunit LMP2 greatly increased in comparison with control and nontolerant animals. Study of accepted grafts showed an increase in the ratio of LMP2/LMP7 immune subunits and 19S/11S regulators in them, compared to the tissue replacing the rejected grafts. Immune proteasomes were almost completely absent from the control intact thyroid tissue, while 19S/11S ratio was maximal in it. Thus, the development of the immune reaction or its suppression are accompanied by a change in the balance between different proteasome forms. Immune subunit LMP7 and 11S regulator are associated with the response against donor tissue. On the contrary, immune subunit LMP2 and 19S regulator are likely to be important for the development of immune tolerance and surviving tissue functioning. Immunofluorescence assay revealed a low content of the immune proteasomes in the follicle cells. Probably, formation of antigens for the major histocompatibility complex class I molecules was impaired by the low content of immune proteasomes, which led to immunological tolerance of hormone-producing follicle cells.     

Exclusion of immune proteasomes from mouse ascitic carcinoma Krebs-II cells

Pools of 26S and 20S proteasomes were studied in the spleen, liver, lung, and ascitic carcinoma Krebs-II of mouse. Western blotting demonstrated that the pool of 26S proteasomes in ascitic carcinoma Krebs-II was twice that in control lung cells and did not significantly differ by total 26S proteasome quantities from the spleen and liver. At the same time, the level of immune subunit LMP7 was 12 times lower in it compared to lung proteasomes and 4–5 times lower compared to spleen and liver proteasomes. Immune subunit LMP2 was undetectable by this technique in the ascitic carcinoma in contrast to the lung, spleen, and liver. All immune subunits in the studied organs and ascitic carcinoma Krebs-II are components of 26S but not 20S proteasomes.     

Ontogeny of Cell-Mediated Immunity of Murine Thymocytes and Spleen Cells

The ontogeny of cell-mediated immunity of spleen cells and thymocytes from B10 mice was studied in both in vitro mixed leukocyte culture and cell-mediated lympholysis reactions. Results show that newborn spleens contain cells competent to respond to X-irradiated allogeneic spleen cells in mixed leukocyte culture. The mixed leukocyte culture response of spleen cells, in terms of both index of stimulation and increment of tritiated thymidine incorporation, seems to be higher for mice four weeks or older than for mice less than four weeks old. The cell-mediated lympholysis response of spleen cells is not detectable until two days postpartum. It reaches adult levels in terms of % cytolysis by day four after birth. Thus, the transition period of the ontogeny of cell-mediated lympholysis response of spleen cells is apparently 0–4 days of age.
Newborn and early postnatal thymocytes (0–7 days of age) respond in mixed leukocyte culture at least as strongly as adult thymocytes (2–3 months of age). The cell-mediated lympholysis response of thymocytes is already detectable at birth, but weaker in terms of % cytolysis when compared with the cell-mediated lympholysis response of thymocytes from two days to six weeks of age. The cell-mediated lympholysis response of thymocytes starts to decline at 6–8 weeks of age. Thus, around the time of birth, there is a transition period in the cell-mediated lympholysis response of thymocytes during which thymocytes start to show high cell-mediated lympholysis reactivity. There is a second transition period between six and eight weeks of age during which the cell-mediated lympholysis response of thymocytes diminishes. The early, as well as late, postnatal cell-mediated lympholysis response of both spleen cells and thymocytes seems to be specific in nature.     

Changes in Proteasome Activity and Subunit Composition during Postnatal Development of Rat

The dynamics of the activities of 26S and 20S proteasomes in the rat liver and spleen have been studied during postnatal development from 1 to 90 days. The activities of proteasome forms both in spleen and in liver increased in adult animals as compared to one day rats. The activities of both proteasome forms in the liver did not differ significantly from those in the spleen at all stages of postnatal development. Using Western blot with monoclonal antibodies to Rpt6 subunit, we confirmed the presence of 26S proteasome in both organs at all stages of postnatal development. Studies with polyclonal antibodies to β1i (LMP2) subunit showed the appearance of the immune subunit in the spleen by day 9 and in the liver only by day 23 of postnatal development. This result suggests the earlier formation of the spleen as an organ with immune functions.__________Translated from Ontogenez, Vol. 36, No. 3, 2005, pp. 205–210.Original Russian Text Copyright © 2005 by Abramova, Astakhova, Sharova.     

Isolation and characterization of bovine thymus multicatalytic proteinase complex

The multicatalytic proteinase complex (MPC or proteasome) from bovine thymus was isolated and purified to homogeneity applying a protocol utilizing ion exchange and gel permeation chromatography as major purification tools. The purified complex shows molecular properties that are common for proteasomal molecules (high molecular mass, multisubunit organization, and multiple proteolytic activities) even though a peculiar subunit composition and the presence of specific regulatory mechanisms affecting the assembled proteolytic activities suggest a specialized function for this complex. Thymus proteasome is characterized by the presence of LMP2, LMP7, and LMP10 (MECL1) subunits, which replace the X, Y, and Z subunits. Since a similar complex was previously isolated in bovine spleen, it appears that the proteasomal population containing the LMP subunits is characteristic for organs involved in immune response. Both the thymus and spleen proteasomes are characterized by a marked efficiency in cleaving peptide bonds after branched-chain and aromatic amino acids, indicating that this proteasomal population is most likely involved in intracellular processing of class I antigenic peptides and is an example of an "in vivo" functioning immunoproteasome. However, in spite of several similarities, the complexes isolated from the two lymphoid organs do not show superimposable functional properties, which suggests the presence of organ-specific regulatory mechanisms affecting each of the proteolytic components assembled in the complex.     

Features of the immune proteasome expression in ascite Zajdela hepatoma after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

The expression of the total proteasome pool, immune subunits LMP2 and LMP7, TAP1 and TAP2 transporters, and RT1A molecules of the major histocompatibility complex (MHC) class I in ascite Zajdela hepatoma cells was studied on the 10th day after implantation into Brattleboro rats with the hereditary defect in the synthesis of arginine-vasopressin (AVP) in the hypothalamus and WAG rats with normal AVP expression. Western-blot analysis revealed a threefold increase in the total number of proteasomes and immune subunit LMP2 and an eightfold increase in the immune subunits LMP7 in Zajdela hepatoma after its implantation in Brattleboro rats as compared with WAG rats. Differences in the expression of immune subunits LMP2 and LMP7 in Zajdela hepatoma in Brattleboro rats may contribute to different functions of these proteasomes, namely, the important role of the subunit LMP7 in antitumor immunity. Zajdela hepatoma growth in WAG rats was accompanied by a fall in both the total proteasome pool and immune proteasomes as compared with their content in Brattleboro rats, whose tumors regressed. The analysis of the content of peptide transporters TAP1 and TAP2 in Zajdela hepatoma implanted into Brattleboro and WAG rats showed their pronounced expression in tumor cells of both rat strains. In Zajdela hepatoma implanted into Brattleboro rats, a threefold increase in the basic molecule of MHC class I-RT1A was identified as compared with its expression in the tumor implanted to WAG rats. Furthermore, the content of CD8 and CD4 T-lymphocytes in the spleen of WAG and Brattleboro rats on the 10th day after implantation of Zajdela hepatoma was analyzed with flow cytometry. An increase in T-lymphocytes expressing the CD8 and CD4 antigens in the spleen of Brattleboro rats after implantation of the tumor as compared with WAG rats was shown. Increased numbers of both cytotoxic T lymphocytes and helper T-cells may facilitate tumor regression in Brattleboro rats. At the same time, a reduced number of subpopulations of T-lymphocytes in the spleen of WAG rats after implantation of hepatoma was accompanied by splenomegaly and growth of the tumor. Based on analysis of the data obtained it can be concluded that the deficiency of AVP in Brattleboro rats in Zajdela hepatoma leads to an increased expression of immune subunit LMP7 and basic molecules of MHC class I resulting in tumor immunogenicity and its elimination by the adaptive immune system.     

Native structure of rat liver immune proteasomes

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αβ activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of “rotated” ubiquitinated proteins.     

Changes in proteasome pool in human papillary thyroid carcinoma development

Searching the antitumor drug targets among proteasomes, “ubiquitous” enzyme systems, may provide a new impulse to the antitumor drug discovery. In this study, changes in the proteasome pool in the development of human papillary thyroid carcinoma were determined. Proteasome activities were evaluated by hydrolysis of commercial fluorogenic peptides. Changes in the expression of the total proteasome pool, proteasome 19S activator and proteolytic constitutive subunits X(β5), Y(β1) and immune subunits LMP7 (β5i) and LMP2 (β1i) were investigated by Western blotting. The distribution of the proteasome subunits in thyroid gland cells was detected by immunohistochemistry. It was shown that the chymotrypsin- and caspase-like activities as well as the expression of the total proteasome pool, proteasome 19S activator and immune subunits increased gradually in the tumors at the T₂N₀M₀ and T₃N₀M₀ stages in comparison with the control tissues. Among the structures studied, the expression of the 19S activator and immune proteasomes, which contain the LMP2 (β1i) subunit, was enhanced to the largest degree in tumor cells. The data obtained may be implicated in a new therapeutic strategy. Taking into consideration the antitumor function of the immune proteasomes, we advance the 19S activator as the target for the development of a novel antitumor therapy.     

Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.     

Interferon-gamma inducible exchanges of 20S proteasome active site subunits: why?

When cells are stimulated with the cytokines IFN-gamma or TNF-alpha, the synthesis of three proteasome subunits LMP2 (beta1i), LMP7 (beta5i), and MECL-1 (beta2i) is induced. These subunits replace the three subunits delta (beta1), MB1 (beta5), and Z (beta2), which bear the catalytically active sites of the proteasome, during proteasome neosynthesis. The cytokine-induced exchanges of three active site subunits of a complex protease is unprecedented in biology and one may expect a strong functional driving force for this system to evolve. These cytokine-induced replacements of proteasome subunits are believed to favour the production of peptide ligands of major histocompatibility complex (MHC) class I molecules for the stimulation of cytotoxic T cells. Although the peptide production by constitutive proteasomes is able to maintain peptide-dependent MHC class I cell surface expression in the absence of LMP2 and LMP7, these subunits were recently shown to be pivotal for the generation or destruction of several unique epitopes. In this review we discuss the recent data on LMP2/LMP7/MECL-1-dependent epitope generation and the functions of each of these subunit exchanges. We propose that these subunit exchanges have evolved not only to optimize class I peptide loading but also to generate LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites which are not proteolytically generated in uninflamed tissues. This difference in epitope generation may serve to better stimulate T cells in the sites of an ongoing immune response and to avoid autoimmunity in uninflamed tissues.     

Beta 2 subunit propeptides influence cooperative proteasome assembly

Vertebrate proteasomes are structurally heterogeneous, consisting of both "constitutive" (or "standard") proteasomes and "immunoproteasomes." Constitutive proteasomes contain three ubiquitously expressed catalytic subunits, Delta (beta 1), Z (beta 2), and X (beta 5), whereas immunoproteasomes contain three interferon-gamma-inducible catalytic subunits, LMP2 (beta 1i), MECL (beta 2i), and LMP7 (beta 5i). We recently have demonstrated that proteasome assembly is biased to promote immunoproteasome homogeneity when both types of catalytic subunits are expressed in the same cell. This cooperative assembly is due in part to differences between the LMP7 (beta 5i) and X (beta 5) propeptides. In the current study we demonstrate that differences between the MECL (beta 2i) and Z (beta2) propeptides also influence cooperative assembly. Specifically, replacing the MECL propeptide with that of Z enables MECL incorporation into otherwise constitutive (Delta(+)/X(+)) proteasomes and facilitates X incorporation into otherwise immunoproteasomes (MECL(+)/LMP2(+)). We also show, using MECL(-/-) mice, that LMP2 incorporation does not require MECL, in contrast with previous suggestions that their incorporation is mutually codependent. These results enable us to refine our model for cooperative proteasome assembly by determining which combinations of inducible and constitutive subunits are favored over others, and we propose a mechanism for how propeptides mediate cooperative assembly.     

Alternative exon usage and processing of the major histocompatibility complex-encoded proteasome subunits.

The finding that two subunits of the proteasome, LMP2 and LMP7, are encoded in the major histocompatibility complex (MHC) has linked the proteasome which represents a major extralysosomal proteolytic system to the processing of intracellular antigens. Here we describe a second form of the human LMP7 cDNA, LMP7-E2, which has been identified during the characterization of novel genes in the MHC. The analysis of the genome organization of LMP7 revealed that LMP7-E1 and LMP7-E2 arise by alternative exon usage. Using specific antibodies against LMP2 and LMP7, we show that they are co-expressed with class I MHC molecules as well as a putative peptide transporter. The polypeptides encoded by LMP7 and LMP2 undergo proteolytic processing when incorporated into proteasomes, and the LMP7 precursor is derived mainly from LMP7-E2. Furthermore, our data suggest that LMP7 and LMP2 are mutually dependent for their incorporation into the proteasomal complex.     

Cell proliferation and differentiation in the fetal and early postnatal mouse thymus

The relationships between cell proliferation and cell differentiation during thymus ontogeny were studied by labeling DNA-synthesizing thymocytes with bromodeoxyuridine and staining with antibodies against CD4, CD8, J11d, phagocytic glycoprotein 1, TCR V beta 8 chain, Thy-1, and IL-2R surface proteins. The development of the thymus was discontinuous, with two well defined growth periods from 13 days to 18 days of fetal life and from 3 days to 6 days after birth, and more progressive growth from day 8 to 2 wk. Cell proliferation started on fetal day 12, 1 day after the arrival of hemopoietic stem cells in the third branchial pouch. These cells were phagocytic glycoprotein 1-positive but IL-2R and Thy-1 negative. Thus, cell proliferation preceded IL-2R expression. Until day 15, CD4-8- thymocytes expanded without differentiation. Then CD4-8+ and CD4+8+ cells appeared; this induction was proliferation dependent and occurred on cells which had already lost IL-2R, but just after maximum expression of this receptor. During several days, the thymus remained of constant size (around 10(7) cells) and behaved like the steady state thymus. On day 3 after birth, expansion started again and was correlated with an increase in CD4-8- proliferation index and IL-2R expression. At the same time, the thymic subset capable of expansion without differentiation was again, transiently, detectable. These results suggest that the inflow of precursor cells into the thymus is permanent but transiently increased at several times during ontogeny. Moreover, the behavior of fetal CD4-8- cells does not appear radically different from that of adult precursors, but the actual difference resides in the variation of the relative proportion of CD4-8- cells at different maturation stages, as revealed by striking variations of IL-2R expression by cycling cells.