ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2014, Vol. 40, No. 1, pp. 36–47. © Pleiades Publishing, Ltd., 2014. Original Russian Text © A.A. Stepanova, Ya.D. Karpova, G.A. Bozhok, V.D. Ustichenko, Yu.V. Lyupina, E.I. Legach, M.S. Vagida, D.B. Kazansky, T.P. Bondarenko, N.P. Sharova, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 1, pp. 42–54.
Proteasomes on Thyroid Tissue Allotransplantation under Induction of DonorSpecific Tolerance in Rats A. A. Stepanovaa, 1, Ya. D. Karpovaa, G. A. Bozhokb, V. D. Ustichenkob, Yu. V. Lyupinaa, E. I. Legachb, M. S. Vagidac, D. B. Kazanskyc, T. P. Bondarenkob, and N. P. Sharovaa a Koltsov
Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119334 Russia Institute for Problems of Cryobiology and Cryomedicine, National Academy of Sciences of Ukraine, ul. Pereyaslavskaya 23, Kharkov, 61015 Ukraine c Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Kashirskoe sh. 23, Moscow, 115478 Russia b
Received April 29, 2013; in final form, June 17, 2013
Abstract—Proteasomes in the liver of August rats (RT1c) were investigated 30 days after allotransplantation of Wistar rat (RT1u) thyroid tissue under renal capsule with/without induction of donorspecific tolerance by donor splenocyte intraportal administration. The levels of total proteasome pool, immune proteasomes con taining subunits LMP2 and/or LMP7, and proteasome regulators 19S and 11S were defined. Intact and shamoperated 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 con trol intact thyroid tissue, while 19S/11S ratio was maximal in it. Thus, the development of the immune reac tion 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 con trary, 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 tol erance of hormoneproducing follicle cells. Keywords: proteasomes, immune proteasomes, portal tolerance, thyroid tissue transplantation, liver, immunolog ical tolerance DOI: 10.1134/S1068162014010105 1
Today, transplantation of endocrine glands is an approach to restoration of the lost hormone produc tion function. However, allotransplantation of organs and tissues activates the recipient’s immune system and triggers acute transplant rejection. As has been shown previously, immunosuppressive drug adminis tration may lead to decrease of hormoneproducing function of endocrine glands [1, 2], therefore, the pos sibility to induce specific tolerance to graft seems promising.
induced. To date, the mechanism of such tolerance is poorly understood; the system of immune response regulation in the liver with immune response– immune tolerance balance shifted towards the latter is considered to play an important role. The DST effect is studied at the cellular level on experimental models, as well as in clinics upon allotransplantation of kidney, heart, intestine, skin, and fragments of peripheral nerves [3–8], however, the molecular mechanisms of the phenomenon are still not clear. Earlier, we were the first to show the prolonged lifetime of endocrine gland grafts upon DST induction by intraportal introduction of splenocytes [9–11]. Morphometric analysis of thy roid tissue graft and analysis of the hormoneproduc ing function demonstrated that induction of tolerance leads to a decrease in the number of cases of acute transplant rejection, allows postponing the chronic
When donor cells are introduced into the recipi ent’s liver portal vein 7–14 days prior to transplanta tion, donorspecific tolerance (DST) to graft may be Abbreviations: DST, donorspecific tolerance; MHC, major his tocompatibility complex; IFN, interferon. 1 Corresponding author: phone: +7 (499) 1358847; fax: +7 (499) 1358012; email:
[email protected].
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rejection of the graft, and increases thyroid tissue pres ervation by 47% 30 days after transplantation [11]. Multifunctional enzyme system, proteasome, may play an important role in the development of portal vein tolerance. One of the factors supporting the hypothesis is the successful application of proteasome activity inhibitor bortezomib as an immunosuppres sant in transplantology [12–14]. Proteasomes are large protein complexes performing selective degrada tion of cellular proteins [15, 16]. Multiple forms of proteasomes in mammalian cells differ by proteolytic subunits and associated regulators. The 19S regulator functions in recognition and binding of ubiquitinated proteins, their unfolding, and pulling to the pro teolytic core. The 11S regulator acts as a proteasome adapter. Besides, generation of antigen epitopes of some proteins for the major histocompatibility com plex (MHC) class I is intensified in the presence of 11S. Immune proteasomes are referred to a special group of proteasomes containing proteolytic subunits LMP7 (β5i), LMP2 (β1i), and LMP10 (MECL1, β2i) instead of proteolytic subunits X(β5), Y(β1), and Z(β2), as in constitutive proteasomes, and participat ing in the formation of antigenic determinants for pre sentation within MHC I molecules [17]. Therefore, immune proteasomes are necessary for development of complete Tcell immune response. Involvement of immune proteasomes in the functioning of Tlympho cytes is not limited to formation of antigen epitopes. Immune proteasomes are important in survival, pro liferation, and activation of lymphocytes in immune response [18]. In case of deficiency of immune subunit LMP2, effective activation of transcription factor NFκB is impossible [19, 20]. The specific function of immune proteasomes not related to their immune properties, that is involvement in suppression of oxi dative stress, has been reported [19]. Nevertheless, there are no data on participation of proteasomes (including immune proteasomes) in the development of specific immune tolerance. At the cellular level, major processes of DST devel opment are localized in liver. Despite the fact that liver is a secondary lymphoid organ, lymphocyte activation processes may occur in the liver [21]. However, lym phocytes activated in liver do not gain the effector antiinflammatory phenotype, but, on the contrary, become regulatory tolerogenic cells. In general, sev eral specific features of liver immunity, important for maintenance of its special status of immune tolerance domination over immune response, may be distin guished [22, 23]. Firstly, these include specific features of professional and nonprofessional antigenpresent ing liver cells [21, 24–26]; secondly, unique cytokine profile of the microenvironment [27]; thirdly, high efficient nonspecific immune protection allowing for the triggering of a specific response in “extreme” cases; and lastly, the deletion of activated antiinflam RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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matory Tlymphocytes [22]. These mechanisms are considered to support the state of immune tolerance to allografts. In the work, we aimed to find out changes occur ring at different proteasome forms expression and localization in liver and thyroid graft of rats with allo geneic thyroid tissue transplantation upon induction of DST. RESULTS AND DISCUSSION The study was performed on liver of recipient rats 30 days after thyroid tissue transplantation. Experi mental animals were divided into two groups. The first group received donor splenocytes in their portal vein 7 days before transplantation, while the second group was not subjected to the procedure. Control groups consisted of intact and shamoperated animals. This model of experiment allows us to reveal the mecha nisms of portal vein tolerance. Functional grafts with retained structure, tissue, replacing the rejected grafts, and an intact thyroid gland were analyzed. Proteasome subunits in rat liver preparations with and without induction of DST. Subunits of various pro teasome forms in cleared liver homogenates of control and experimental animal groups were studied by West ern blotting. The level of expression of αsubunits (α1,2,3,5,6,7), present on all proteasome forms, reflects expression of total pool of proteasome. The level of expression of immune subunits LMP2 and LMP7 determines the level of immune proteasomes containing subunits LMP2 and/or LMP7 respectively. Expression of LMP10 (MECL1) was assessed by the expression of LMP2 subunit since, as a rule, they incorporate proteasome jointly. Subunit LMP7 may incorporate proteasomes both with subunits LMP2 and LMP10 (MECL1) or without them [28]. Subunits Rpt6 and PA28α are markers of 19S and 11S proteasome regulators, respectively. Figures 1 and 2 demonstrate the levels of immune proteasomes, the total pool of proteasomes, and regulators 19S and 11S. In livers of rats of both groups with grafts, the level of immune proteasomes with subunits LMP2 and/or LMP7 increased while total proteasome level decreased (Fig. 1), also the level of 19S regulator decreased and that of the 11S level increased if com pared to control groups (Fig. 2). It should be noted that proteasome pool in liver of shamoperated rats did not differ from that of the intact rats by the con trolled parameters. So, figures 1 and 2 report only the data for the liver of intact rats. Between the groups of experimental animals with and without induction of DST, significant difference in the expression of Rpt6 subunit of 19S proteasome regulator and PA28α subunit of 11S regulator was observed (Fig. 2). The ratio between the expression of these regulators in livers of control animals was set as Vol. 40
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Fig. 1. Expression of subunits α1,2,3,5,6,7 and immune subunits LMP2 and LMP7 of proteasomes in rat liver after transplanta tion of thyroid tissue. (a) Western blotting of proteins from cleared rat liver homogenates using antibodies to subunits α1,2,3,5,6,7 and immune subunits LMP2 and LMP7. (b) Relative values of integral absorption of protein bands normalized against βactin levels; the value in control group of animals was considered equal to 10 a.u.. “+DST” and “–DST” indicate groups with and with out induction of donorspecific tolerance. Median values are reported, the boxes show interquartile ranges (25–75%), and bars, confidence intervals at significance level α = 0.05, n ≥ 5. * p < 0.01.
the control value (100 a.u.). No significant differences were identified in other proteasome forms. These data may evidence the following processes in the liver. After the introduction of a graft, the fraction of immune proteasomes in the total pool increases and the pattern of the proteasome regulator expression changes. This indicates the switch of the total protea some pool functions to graft rejection reactions or development of tolerance to it. Earlier, it was proven that during the process of allogenic tumor graft rejec tion, at the peak of the response in the liver accumu lated functional cytotoxic lymphocytes actively pro duced IFNγ [29]. This may explain the increased immune proteasome subunits expression in liver upon rejection of thyroid tissue grafts, since IFNγ is one of the factors inducing expression of immune subunits of proteasomes in the process of immune response [17]. A high content of 19S regulator in liver of control groups of animals indicates that normally selective degradation of ubiquitinated proteins in liver is not associated with either active immune response or intensive development of immune tolerance. In ani mals with DST induced, the 19S/11S ratio decreased mainly due to an increase in the amount of 11S activa tor.
It is known that proteasomes with the same com position of proteolytic subunits may differ consider ably depending on the interaction with different regu lators. There is no clarity on the physiologic functions of 11S proteasome activator today. It has been found that 11S activator, similar to immune subunits of pro teasomes, may be involved in the immune response induced by IFNγ [30–33]. On the one hand, in a num ber of cases 11S activator promotes generation of pep tides for MHC I [31, 33], on the other, association of 11S activation with peptide transporter TAP increases the efficiency of the peptides transfer into endoplas mic reticulum where they bind with MHC I molecules [34, 35]. Besides, immune proteasomes with the regu latory complex have been shown to participate in the suppression of oxidative stress [36]. Therefore, the results suggest that, besides these known functions in the immune response, 11S regulator of proteasomes may be involved in the development of immunological tolerance. Its precise role in the process requires fur ther investigation. Specific features in immune proteasome distribution in hepatocytes of rats with and without the induction of DST. To reveal the specific localization of proteasomes at cellular level, LMP2 and LMP7 proteasome subunits were detected by immunohistochemistry in ultrathin
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Fig. 2. Expression of proteasome regulators in rat liver after thyroid tissue transplantation. (a) Western blotting of proteins from cleared rat liver homogenates using antibodies to Rpt6 subunit of 19S regulator and subunit PA28α of 11S regulator. (b) Relative values of integral absorption of protein bands normalized against βactin levels; the value in control group of animals was consid ered equal to 10 a.u. On the right, ratio between proteasome regulator expression levels 19S/11S is reported. See caption to Fig. 1 for designations. Statistical differences at p < 0.01 (*) and p < 0.05(О) are shown.
sections of liver (Fig. 3). As it has been found by immunoblotting, in control tissue immune protea somes are expressed at lower level, which is supported by immunohistochemistry results. In the group of ani mals with induced DST, in hepatocytes the redistribu tion of proteasomes occurs at the level of cells, they are compartmentalized. Association of proteasomes with cellular structures (components of cytoskeleton, membranes of endoplasmic reticulum, etc.) of the kind may be important for the functioning of hepato cytes, which are involved in the development of portal tolerance [37]. Immune proteasome content in liver mononuclear cells. Major participants of induction of portal toler ance and its maintenance are professional and non professional antigenpresenting cells, lymphocytes, monocytes, and other cells comprising the small pop ulation of liver mononuclear cells [24–26]. Immune proteasome content in liver mononuclear cells was determined by flow cytometry. In animals with induced DST and accepted grafts, the amount of cells expressing immune subunit LMP2 of proteasomes considerably increased if compared to the animals without induced DST or control animals (67.8, 11.6, and 16.4% of LMP2containing cells respectively) (Fig. 4). No statistically significant differences in the number of cells containing LMP7 immune subunit was observed among the experimental groups. There RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
fore, there are differences in expression of immune proteasome forms containing subunit LMP2 and those containing subunit LMP7 in response to toler ance induction, which evidences the difference in functions they execute. The results are promising for further studies. It is important to find out which pro cesses determine the important shift in the content of immune proteasomes with subunit LMP2: is it the change in the composition of mononuclear cell popu lation or the change in expression of immune subunit LMP2 in a single type of cells. Histology analysis of thyroid tissue grafts and thy roxine content in rat blood. The rate of graft survival was evaluated by histological analysis and the level of thyroid hormones produced. These analyses suggest a conclusion about whether the graft morphology is typ ical of the thyroid tissue or not, and evaluation of the hormoneproducing function of the graft. In the blood serum of animals total thyroxine was determined 30 days after transplantation. Two sub groups of animals were isolated: one with low and another with high thyroxine content (mean values of 23 and 60% in content in control animals, p < 0.01). The first subgroup encompassed all animals without DST induced and two animals with DST. Three rats with DST were referred to the subgroup with high thy roxine content. The increase in the total thyroxine in rat blood evidences functioning thyroid tissue graft. Vol. 40
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Fig. 3. Immunohistochemical labeling of cells in ultrathin sections of liver with antibodies against subunit LMP2 (a–c) and LMP7 (d–f) and secondary fluorescent antibodies. (a, b, d, e) Rat liver after transplantation; (a, d) rat liver without the induction of DST; (b, e) rat liver with the induction of DST; (c, f) liver of control animals. Scale bar corresponds to 50 μm.
Histology analysis revealed signs of graft rejection in animals with low total thiroxine level. Complete degradation of follicular structure of thyroid tissue and its substitution with connective tissue occurred (Fig. 5). Differences in morphometric parameters of tissues replacing the rejected grafts were revealed between groups with and without the induction of DST. In ani mals with DST, the amount of connective tissue was insignificant, with the ratio between the volume of connective tissue to the volume of initially grafted tis sue being below 1. In samples recovered instead of grafts in animals with induced DST only connective tissue is present, which is characteristic of the end of the immune rejection process. Nevertheless, these samples are larger in size and the ratio of recovered tis sue volume to the volume of initially grafted tissue exceeds 1. Grafts in animals with increased thyroxine blood levels exhibit a different structure (Fig. 5). Part of the graft tissue is represented by connective tissue, how ever there are regions with preserved structure of thy roid tissue, that is, follicles lined with pyramidal thy rocytes, some of which contain resorption vacuoles of colloid, which evidences an active hormoneproduc ing function. Small vessels penetrate the graft tissue, which indicates a successful vascularization process and, consequently, graft supply with nutrients.
Proteasomes in the graft. What changes were observed at the level of different proteasome forms in surviving grafts and tissue replacing the rejected grafts? No differences in the expression of the total pool of proteasomes has been observed among grafts and between the grafts and the control thyroid tissue. Immune proteasomes containing subunits LMP2 and/or LMP7 are little expressed in control thyroid tissue, while in animals with thyroidectomy followed by thyroid tissue transplantation, a complex picture of immune subunits expression was noted (Fig. 6). To visualize the data, the ratio between the expres sion of immune subunits LMP2 and LMP7 was calcu lated. The normal level (10 a.u.) was chosen to be the ratio of the expression of the subunits in tissue replac ing one of the rejected grafts, since no comparison could be made with intact control thyroid tissue due to the extremely low expression of the LMP7 subunit in it. As follows from Fig. 6, in accepted grafts (with developed follicular structure) the ratio exceeds con siderably that in the tissue replacing the rejected grafts. The results coincide with our data published earlier [38]. When studying grafts of ovaries in a similar experimental setup we found enrichment of surviving grafts with LMP2subunit immune proteasomes, and the rejected grafts, with LMP7subunit immune pro teasomes [38]. It may be assumed that immune sub unit LMP7 is involved in providing an efficient
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Fig. 4. Cytofluorimetry analysis of expression of LMP2 and LMP7 subunits in rat liver mononuclear cells. (a) In the left panel, dot plot of forward and side scattering of liver mononuclear cells is presented; the ovals highlight two cell populations. In the right panel, relevant histograms of LMP2 and LMP7 subunit expression in both populations of liver mononuclear cells of three groups of animals are presented. Representative data of individual experiments are presented. (b) Percentage of mononuclear cells express ing subunits LMP2 and LMP7 in rat liver of animal groups under study. Total numbers of LMP7+ and LMP2+ cells in both isolated populations are reported. Median value and confidence interval at confidence level α = 0.05 are indicated; * p < 0.01.
immune response against foreign tissue, while immune subunit LMP2 accompanies graft acceptance and, most likely, plays other roles as well. Its function may be connected with nonimmune reactions, for example, oxidative stress suppression [35, 39] or other processes. In samples of thyroid tissue, the levels of expression of Rpt6 and PA28α subunits comprising 19S and 11S regulators, respectively, also change if compared to control (Fig. 6). In control samples of thyroid tissue the level of 19S regulator expression was higher than in all graft samples and the level of 11S regulator expres RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
sion was the lowest. In grafts of different groups, 11S regulator expression was increased and that of 19S reg ulator, decreased, to a different extent. Figure 6 dem onstrates the result of the calculation of the ratio between 19S regulator expression (by the expression of Ppt6 subunit) to the level of 11S regulator expression (by the expression of PA28α subunit). The ratio between the expression of these regulators in control thyroid tissue was considered normal (100 a.u.). The value of the ratio of proteasome regulators’ expression correlates well with the functional state of the grafts: the higher the quality of graft acceptance, the more the Vol. 40
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Fig. 5. Histology preparations of thyroid tissue, hematoxylin and eosin staining. (a, b) Tissue samples observed upon graft rejec tion; (c, d) surviving grafts in rats with high level of blood thyroxine; and (e, f) intact control, thyroid tissue; 1, graft or tissue replacing the graft; 2, kidney tissue; and 3, thyroid tissue. Scale bar corresponds to 100 μm.
ratio shifts to the expression of 19S regulatory com plex. On the contrary, in tissue replacing the graft in the case of its rejection, expression 11S proteasome regulator, which may be involved in the immune response at the stage of generation of antigen determi nants, is activated [40]. Specific features of proteasome subunit distribution at the cellular level in thyroid tissue. To reveal the spe cific features of LMP2 and LMP7 proteasome distri bution at cellular level, cells in sections were stained with antibodies against the subunits (Fig. 7). In grafts with no follicle structure no difference in immune proteasome distribution between cells was observed. In control thyroid tissue in cells lining the follicles immune proteasomes were not detected. In cases of thyroid tissue graft acceptance, we observed a specific distribution of cells expressing
immune proteasomes. Subunits LMP2 and LMP7 were revealed in cells surrounding the follicle struc ture, while follicular cells, thyrocytes, similarly to cells of control thyroid tissue, hardly express immune pro teasomes (Fig. 7). This might indicate the absence of an immune response against hormoneproducing cells, since the formation of antigen epitopes for MHC I molecules is hindered in their cytoplasm. This phe nomenon may have two explanations: either the potency of thyrocytes to express immune proteasomes is decreased, or the required signals—proinflamma tory cytokines necessary for Tcell mediated immune response—are absent. Not only thyrocytes lining the follicles, but also cells surrounding the follicles are of particular interest. The difference in content of LMP2 and LMP7con taining immune proteasomes in some cells was
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Fig. 6. Content of proteasome subunits and regulators in rat thyroid gland grafts. (a) Western blots of protein isolated form clari fied homogenates of grafts and intact thyroid tissue using antibodies to immune subunits LMP2 and LMP7 of proteasomes and subunits Rpt6 and PA28α of proteasome regulators. (b) Ratio between levels of expression of immune subunits LMP2/LMP7 (the value in tissue replacing one of the rejected grafts is considered 1) and subunits Rpt6/PA28α (value in intact thyroid tissue is con sidered 100). I, tissue replacing the rejected graft; II, accepted graft; C, intact thyroid tissue. Median value and confidence interval at confidence level α = 0.05 are indicated; * p < 0.01.
detected (Fig. 7). In certain cells, immune protea somes with subunit LMP7 are mainly detected while in the others, those with subunit LMP2. Also, cells containing both immune subunits were found. The results explain the difference between these subunits detected by immunoblotting. A question arises on whether all studied cells are graftinfiltrating lympho cytes. Investigation into what are these cell types and what are the reasons for the difference in immune pro teasome subunit expression is the essential part of our further studies. One may not exclude the possibility that some cells expressing immune proteasomes are responsible for the development of tolerance to the graft. On the one hand, these cells somehow may pro tect hormoneproducing cells from the attack of the immune system, on the other, probably, the stroma cells are involved in the fight with oxidative stress, which often becomes the initial stage of the graft rejec tion process [41, 42]. Besides, the tissue may provide mechanical support to thyrocytes, since it has been shown that a threedimensional scaffold is required for their survival and normal functioning [43]. Therefore, the fact that proteasomes play a certain role in the development of immune response to the graft and tolerance to the allograft is undoubted. The development of the immune response or its suppres sion depends on the balance of various forms of pro teasomes in graft tissue. The shift of proteasome pool toward forms with immune subunit LMP7 and regula tor 11S characterizes the progression of the immune response, while a shift towards forms with immune subunit LMP2 and regulator 19S, the progression of immune tolerance. Besides, proteasomes containing immune subunit LMP2 in liver mononuclear cells, apparently, are involved in the progression of immune tolerance. Therefore, the work revealed a new nonim RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
mune function of immune proteasomes containing the LMP2 subunit. Probably, the increased content of LMP2 subunit in liver cells may be a marker for sur vival of the graft. EXPERIMENTAL Treatment of animals. Procedures on animals were performed by the staff of the Institute for Problems of Cryobiology and Cryomedicine of the National Acad emy of Sciences of Ukraine according to the List of Ethical Principles for Experiments on Laboratory Ani mals. Experiments were performed on 3–4 monthold male rats of Wistar and August lines. Wistar rats (RT1u) were donors and August rats (RT1c), recipients. To induce DST, animals received 1 mL of physio logical saline containing 1 × 107 splenocytes into the liver portal vein 7 days prior to transplantation. Sple nocytes were obtained from spleen of Wistar rats. Thy roidectomy in August rats was performed 7 days prior to transplantation. The thyroid gland was taken from Wistar rats, divided into four fragments, washed 3– 4 times in saline, and grafted under the renal capsule in the Augustline recipients [10]. The dose of trans planted material was 35–40 mg. On day 30 after trans plantation, several groups of animals were studied: group 1, intact control (3 rats), group 2, animals with intraportal administration of splenocytes followed by transplantation of thyroid tissue under the renal cap sule (5 rats), and group 3, animals with thyroid tissue transplantation without preliminary induction of DST (5 rats); a single group of shamoperated animals was used in a study on ovary transplantation [38] and thy roid tissue transplantation. Antibodies. Rabbit polyclonal antibodies (pAbs) to proteasome subunits LMP7 and LMP2, mouse mon Vol. 40
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oclonal antibodies (mAbs) to LMP2 proteasome sub unit and Rpt6 subunit of the 19S regulator of protea somes, combined mouse mAbs to α1,2,3,5,6,7 sub units, rabbit pAbs to PA28α subunit of the 11S regulator (Biomol, Great Britain); mouse mAbs to β actin (Santa Cruz, United Stated); Alexa488conju gated antirabbit IgG Abs and Alexa546conjugated antimouse IgG Abs (Invitrogen, United States); PE conjugated antimouse IgG Abs (BD Biosciences, United States); and peroxidaselabeled antimouse IgG and antirabbit IgG Abs (Amersham Biosciences, Great Britain). Determination of thyroid hormones in blood serum of the animals. To evaluate the hormoneproducing function of thyroid tissue grafts, the level of thyroxine in blood serum was determined. Total thyroxine (free and bound to transporter proteins) was determined by radioimmunoassay using a TOTAL T4RIA KIT (Immunotech). Flow cytometric analysis of liver mononuclear cells. Rat liver was perfused with calciumfree buffer (0.1 M PBS, pH 7.4, 5 mM EDTA) through the portal vein for 5 min. Then, the liver was isolated and perfused with buffer containing 0.1 M PBS, 0.4 mg/mL collagenase type IV (Sigma, United States), 5 mM СаСl2, 25 ng/mL DNase I, 5 mM MgCl2, at 37°С for 10 min, minced with scissors, incubated in buffer with collage nase at 37°С for 30 min and centrifuged at 20 g and 4°С for 2 min. Cells were washed in the supernatant of the collagenase and centrifuged in Percoll, 30–80% density gradient, at 4°С and 400 g for 30 min. Mono nuclear cells were collected from the Percoll density interface and washed two times with 0.1 M PBS at 4°С. The viability of isolated cells was verified by supravital staining with tripan blue and was approxi mately 86%. Isolated mononuclear cells were fixed in 2% paraformaldehyde for 15 min, permeabilized in 1% saponin solution in 0.1 M PBS for 15 min at a con centration of 1 × 107 cells per test. Cells were incu bated with rabbit pAbs to LMP7 and mouse mAbs to LMP2 (1 : 500) in 0.1 M PBS containing 1% BSA overnight at 4°С. After washing, cells were incubated with Alexa488labeled antirabbit IgG and PElabeled antimouse Abs (1 : 500) for 30 min at room tempera ture. Background was determined by incubation of liver cells with secondary antibodies. Cell fluorescence was measured on a FACSCanto II (BD Biosciences, United States). Histology analysis and immunofluorescence labeling of cells in sections of liver, thyroid, and graft tissue. Tis sue samples (liver, thyroid, or graft tissue) were washed several times with 10 mM PBS and fixed for 2 h at room temperature in 4% paraformaldehyde. Tissue samples were maintained overnight in 30% sucrose at 4°C for cryoprotection. Then, tissue samples were fro zen in nhexane at –30°C. Serial 8–12μm thick sec tions were immobilized on polylysine glass. For histol ogy analysis of the graft state, hematoxylin and eosin
staining was used. Glasses were incubated for 5 min in hematoxylin, washed with water several times, incu bated 3 min in eosin, washed with distilled water, and embedded in a VitroGel medium. To study the distribution of immune subunits of proteasomes over cells, immunohistochemical stain ing of 10μm sections was used. To block nonspecific binding of antibodies and increase the availability of antigens, glasses were incubated in blocking solution (3% normal goat serum (NGS), 0.3% Triton X100, and 10mM PBS) for 30 min at room temperature. Incubation with primary antibodies to LMP7 and LMP2 subunits (1 : 400) was performed overnight at room temperature. Glasses were washed several times with 10 mM PBS and incubated with secondary fluo rescently labeled antibodies to rabbit IgG (Alexa 488) and mouse IgG (Alexa 546) (1 : 600) in darkness for 1 h at room temperature. Samples were embedded in Mowioll mounting medium, images were recorded using a Leica TCS SP5 STED confocal microscope of the Center for collective use “Optical Research Group” of the Koltsov Institute of Developmental Biology, Russian Academy of Sciences. Preparation of cleared homogenates and immunob lotting. Liver, thyroid, and graft tissues were washed with 10 mM PBS, weighed wet, frozen, and stored at –70°C. Cleared homogenates were prepared at 0– 4°C. Three volumes of extracting buffer (50 mM Tris HCl, pH 7.5, 10 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10 mM Na2S2O5, 0.5 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 μg/mL aprotinin) were added to the sample of thawed tissue and homoge nized in microcentrifuge tubes, first with a teflon pes tle and then in an UltraTurrax T 10 basic (IKA WERKE) homogenizer. The homogenates were cen trifuged for 30 min at 15400 g and 4°C. Supernatant (cleared homogenate) was used for further studies. Electrophoresis was performed according to Laemmli [44]. Samples were loaded in the amount of 50–60 μg protein per lane, commercial Fermen tasPageRulerTM was used as a molecular weight marker. Protein transfer onto a nitrocellulose membrane was performed for 1–1.5 h at 100 V in transfer buffer (25 mM Tris, 192 mM glycine, 20% ethanol). The quality of transfer was evaluated with Ponceau C stain ing. Membranes were washed free of the dye in TNT (10 mM TrisHCl, pH 7.4, 150 mM NaCl, and 0.1% Tween20). To prevent nonspecific sorption of antibod ies, the membrane was blocked with 5% milk in TNT. Incubation with primary antibodies to proteasome subunits, proteasome activators (1 : 1500), and βactin (1 : 400) were performed for 2 h at room temperature or overnight at 4°C. Then, membranes were washed several times with TNT. Incubation with secondary antibodies to mouse or rabbit IgG (1 : 1500) was per formed for 1 h at room temperature. After incubation with secondary antibodies, membranes were washed
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Fig. 7. Immunohistochemistry of ultrathin sections of grafts using antibodies to LMP2 (a, c, e, g) and LMP7 (b, d, f, h) subunits and secondary fluorescent antibodies. (a–d) Accepted grafts; (e, f) samples of tissue replacing a rejected graft; (g, h) intact thyroid tissue. Arrows indicate cells containing primarily subunit LMP7 or subunit LMP2. Scale bar corresponds to 50 μm. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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several times with TNT and developed with a standard method of enhanced chemiluminescence (ECL) with an Amersham ECL Plus™ WesternBlottingDetection Reagents kit. Xray films were scanned and the images were pro cessed using ImageJ software evaluating the integral absorption of protein bands. The results on the levels of proteasome subunit and activator expression were normalized against βactin levels. Statistical analysis. Statistical significance was determined by using nonparametric Mann–Whitney U test. The Statistica7 software package was used for statistical data processing.
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