Bergant Marušič, Martina

Griffiths, Gareth

Jeras, Matjaž

Zavašnik-Bergant, Tina

English

Digital repository of Slovenian research organizations

IntroductionFollowing the processes of internaliza-tion, different particles, parts of apoptotic and necrotic cells, pathogens (bacteria, viruses) and other foreign proteins are taken up into the endocytic pathway of immune cells such as macrophages and dendritic cells (DC).1 As antigen-present-ing cells (APC) these cells play a pivotal role in the adaptive immunity by initia-tion of efficient T-cell mediated immune response.2 DC circulate through tissues like veiled scavengers. Their maturation Radiol Oncol 2006; 40(3): 183-8.Different localisation of cystatin C in immature and mature dendritic cellsTina Zavašnik-Bergant1, Martina Bergant2, Matjaž Jeras2, Gareth Griffiths31Jožef Stefan Institute, Dept. of Biochemistry and Molecular Biology, Ljubljana, Slovenia; 2Blood Transfusion Centre of Slovenia, Tissue Typing Centre, Ljubljana, Slovenia; 3European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, GermanyBackground. Limited antigen degradation by proteolytic enzymes and their control by protease inhibitors represent a crucial step in generating antigenic peptides inside the endocytic pathway of antigen-presenting cells such as dendritic cells. Methods. Human dendritic cells were used as a cell model in which quantitative immunogold electron microscopy was applied in order to study endogenous protease inhibitor cystatin C. Ultrathin cryosec-tions were prepared from immature and mature dendritic cells and labelled with specific antibody. Under the transmission electron microscope gold particles, bound to specific probe (antibody), pointed the exact localization of labelled inhibitor. Results. Quantification of immunogold labelling and further statistical analysis by chi-squared test empha-sized the differences in cystatin C content in different cell compartments. Conclusions. Statistically significant differences in intracellular distribution of cystatin C have been deter-mined between immature and mature dendritic cell population.Key words: dendritic cells; cryoultramicrotomy; microscopy, electron; immunohistochemistryReceived 1 June 2006Accepted 21 July 2006Correspondence to: Tina Zavašnik-Bergant, Department of Biochemistry and Molecular Biology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia. Tel.: +386 1 477 3474; Fax: +386 1 477 3984; E-mail: tina.zavasnik@ijs.siThe paper was presented at the 4th Conference on Experimental and Translational Oncology (Kranjska gora, Slovenia, March 22-26, 2006) as invited lecture.Abbreviations: Ag – antigen, APC – antigen-pre-senting cells, DC – dendritic cells, MHC II – major histocompatibility complex class II molecules, TNF-α– tumour necrosis factor-alpha, TEM – transmission electron microscopyoccurs as they migrate from peripheral tis-sues, where they search for antigen (Ag), to lymphoid organs, where they present captured and processed Ag to specific T cell receptors. Limited Ag degradation performed by proteolytic enzymes (cathe-psins) and their control by intracellular protease inhibitors represent a crucial step in generating antigenic peptides.3,4Due to the increased research interest in events taking place inside the endocytic pathway5 of immune cells, a high-resolu-tion technique has been applied to observe cell organelles and search for specific intra-cellular proteins. Differentiation of mono-cytes to immature DC and their further maturation with TNF-α to mature DC pop-ulation6,7 was applied as a model system for studying transport pathway of endog-enous protease inhibitor cystatin C in im-mune cells. Described method consists of preparation of ultrathin cryosections from frozen cells,8 their immunogold labelling,9 quantification of attached gold particles by transmission electron microscopy (TEM) and statistical analysis of obtained data.10Material and methodsImmature DC were generated from isolated blood monocytes and further stimulated with TNF-α as described.6,7 Cells were fixed with 4% paraformaldehyde (Sigma –Aldrich). They were embedded into 10% gelatin (Merck), infused with 2.3 M sucrose (Merck) and cut to ultrathin cryosections (70 – 80 nm) at minus 120 °C8,9 using Leica EM FC6 ultramicrotome for cryosectioning. Sections were thawed in a droplet of 1:1 mixture of 2.3 M sucrose (Merck) and 2% methyl cellulose (Sigma-Aldrich). Retrieved ultrathin sections were put on carbon-coated formvar films (TAAB Lab.) and spread over hexagonal Cu/Pd grids (Agar). Sections were labelled with rabbit anti-hu-man cystatin C antibody11 or rabbit anti-hu-man cathepsin S antibody.12 Their specifici-ty and cross-reactivity against recombinant human antigens 13,14 was checked as report-ed 7,11,12,15 prior to described immunogold labelling experiments. After the removal of an excess of primary antibody thin sections of DC were incubated with Protein A–gold conjugate with 10-nm gold particles (Cell Microscopy Centre, University Medical Centre Utrecth). Sections were contrasted with uranyl acetate (Aurion) and viewed with Philips CM120 BioTWIN transmis-sion electron microscope. KeenView digital camera and iTEM software were used for taking pictures. Quantification of immunogold labelling patterns in the same sets of compartments (or organelles) in different groups of cells (immature and mature DC) was performed for protease inhibitor cystatin C. Random sampling of specimen was followed by counting of approximately 200 gold par-ticles on two labelled grids. The distribu-tions of gold particles in different compart-ments was compared by contingency table analysis with statistical degrees of freedom for chi-squared values being determined by the number of compartments and the number of experimental groups of cells. Compartmental chi-squared values making substantial contributions to the total chi-squared values identified where the main differences between groups resided.10ResultsStrong immunogold labelling of Golgi was observed in immature DC (Figure 1) where-as in mature DC Golgi remained practically unlabelled after incubation with specific antibody (Figure 2). Gold counts and re-sults of calculations for Golgi and other compartments (organelles) are provided in Figure 3 and Table 1. High number of gold Zavašnik T et al. / Cystatin C in human dendritic cells184Radiol Oncol 2006; 40(3): 183-8.particles associated with Golgi (Figure 1, Figure 3A), endoplasmic reticulum (Figure 3A) and nuclear membrane (Figure 3A) indicated high expression of cystatin C in immature DC. In mature DC number of gold particles increased in multivesicular bodies and small vesicles near the plasma membrane (Figure 3B).Of 204 gold particles counted in im-mature DC 59 were associated with Golgi (Figure 3A, Table 1). In contrast, in mature DC 203 gold particles were distributed so that only 2 were observed on Golgi (Figure 3B, Table 1). By contingency table analysis, the predicted number of gold particles on a given compartment in a given cell group is given by [(column total × row total)/grand total] where the grand total is the sum of the column (or row) totals.10 Consequently, the predicted number of Golgi gold parti-cles in immature DC, for example, is calcu-lated to be [(61x204)/407] or roughly 30.6 (Table 1). For each compartment and cell type, the partial chi-squared value is given Zavašnik T et al. / Cystatin C in human dendritic cells 185Radiol Oncol 2006; 40(3): 183-8.Figure 2. Golgi apparatus in human mature dendritic cells is not labelled for cystatin C. Bar: 200 nm.Figure 1. Strong immunogold labelling of protease inhibitor cystatin C in Golgi apparatus of immature dendritic cells. Gold particles are showing the exact localization of cystatin C. Bar: 200 nm.Figure 3. Comparison of immunogold labelling of inhibitor cystatin C in immature (A) and mature (B) den-dritic cells. Gold particles were counted on two grids. Distributions of app. 200 gold particles (+ SD) are shown. Compartments: (a) nucleus, (b) nuclear membrane, (c) rough endoplasmic reticulum, (d) Golgi apparatus, (e) small vesicles, (f) multivesicular bodies, (g) multilamelar bodies, (h) vesicles near plasma membrane, (i) plasma mem-brane, (j) cytoplasm, (k) mitochondria, (l) unknown structures, other. by [(observed golds − expected golds)2 / expected golds].10 Therefore, the partial chi-squared value for Golgi in immature DC is equivalent to [(59 − 30.6)2/30.6] or 26.4. Table 1 shows that the total chi-squared value is 120.02. For 11 degrees of freedom [(2 − 1 columns) by (12 − 1 rows)] this indicates that the null hypothesis of no difference between groups must be rejected (P < 0.001). Therefore, the distribution of cystatin C is significantly different in immature and mature DC and the gold counts are consist-ent with a shift towards greater-than-pre-dicted labelling of Golgi in immature DC (Table 1).Furthermore, the partial (or compart-mental) chi-squared values indicate that immature DC have more-than-expected gold particles on Golgi, endoplasmic re-ticulum and nuclear membrane (Table 1). On the contrary, partial chi-squared val-ues indicate that mature DC have fewer-than-expected gold particles on Golgi but more-than-expected gold particles on the vesicles near the plasma membrane (within a distance of 200 nm), small vesicles and multivesicular bodies (Table 1).Population of multivesicular bodies (in addition to some other organelles) has also been labelled with anti-cathepsin S antibody and gold in both, immature and mature, DC population (data not shown).DiscussionAntigen processing by MHC class II mol-ecules (MHC II) is tightly linked to the ac-tivity and stability of proteolytic enzymes present inside the endocytic pathway of DC.16 Lysosomal cysteine proteases (cathe-psins), which constitute a major portion of this proteolytic system, have an essential role in both Ag processing and matura-tion of MHC II.17 Among the regulatory molecules of cathepsins a low molecular weight (MW 13.000) type II inhibitor cysta-tin C was suggested to have a role in con-trolling the proteolytic activity of cathepsin S inside the endocytic route of mouse DC.18,19 By binding to cathepsin S during Zavašnik T et al. / Cystatin C in human dendritic cells186Radiol Oncol 2006; 40(3): 183-8.Table 1. Labelling distributions of inhibitor cystatin C and chi-squared test. Values represent observed and ex-pected (in brackets) numbers of 10-nm gold particles in immature and mature dendritic cells. For a total chi-squared of 120.02 and 11 degrees of freedom, P < 0.001. These two labelling distributions are significantly different.DC maturation cystatin C was suggested to compromise the step-wise degradation of MHC II-associated chaperone molecule (invariant chain).We have confirmed that in human imma-ture DC cystatin C content was highly el-evated compared to mature DC population as well as to their precursors – monocytes.7 But with this study, cystatin C extremely high content in Golgi apparatus as well as its presence in endoplasmatic reticulum and nuclear membrane, continuous with the endoplasmic reticulum, was quanti-fied for the first time in human immature DC. Furthermore, the transport of cystatin C was shown from Golgi towards the cell membrane, supported by the decrease of cystatin C in Golgi and endoplasmic reticu-lum and increase in labelling of different populations of transport vesicles (small vesicles, vesicles near plasma membrane) and multivesicular bodies.MHC II–loading compartments (multive-sicular bodies) were positive for cathepsin S 13,14 (proposed target enzyme of cystatin C, involved in maturation of MHC II) in both, immature and mature DC (Zavašnik-Bergant, unpublished data). Labelling of multivesicular bodies for cystatin C was higher-than-expected in mature but not immature DC indicating higher content of cystatin C inside Ag-loading compart-ments in mature DC compared to imma-ture population. Differentiation and matu-ration dependence of endogenous cystatin C supports its intracellular regulatory po-tential in human DC7, as well as it further suggests its new role in Golgi apparatus of immature DC.In conclusion, preparation of cryosections of human DC, their immunogold label-ling with specific anti-cystatin C antibody and transmission electron microscopy were successfully applied to quantify protease inhibitor cystatin C in different organelles of immature and mature DC. AcknowledgementsThis work has been performed at EMBL Heidelberg and granted by EMBO fellow-ship (ASTF 90.00-05) to Tina Zavašnik-Bergant. An introduction to the described technique by Uta Haselmann and a support given by Claude Antony (EMBL Electron Microscopy Core Facility) is acknowl-edged.References1. Griffiths G. On vesicles and membrane compart-ments. Protoplasma 1996; 195: 37-58.2. Honey K, Rudensky AY. Lysosomal cysteine pro-teases regulate antigen presentation. Nat Rev Immunol 2003; 3: 472-82.3. Villadangos JA, Schnorrer P, Wilson NS. Control of MHC class II antigen presentation in dendritic cells: a balance between creative and destructive forces. Immunol Rev 2005; 207: 191-205.4. Chapman HA. Endosomal proteases in antigen pre-sentation. Curr Opin Immunol 2006; 18: 78-84.5. Pillay, CS, Elliott E, Dennison C. Endosomal pro-teolysis and its regulation. Biochem J 2002; 263: 417-29.6. Repnik U, Knežević M, Jeras M. Simple and cost-effective isolation of monocytes from buffy coats. J Immunol Methods 2003; 278: 283-92.7. Zavašnik-Bergant T, Repnik U, Schweiger A, Romih R, Jeras M, Turk V, et al. Differentiation- and maturation-dependent content, localization and secretion of cystatin C in human dendritic cells. J Leukoc Biol 2005; 78: 122-34.8. Tokuyasu KT. A study of positive staining of ul-trathin frozen sections. J Ultrastruct Res 1978; 63: 287-307.9. Tokuyasu KT. Immunocytochemistry on ultrathin cryosections. In: Specer DL, Goodman RD, Leinwand LA, editors. Cells, a laboratory manual. Vol. 3. Subcellular localization of genes and their products. Cold Spring Harbour: Laboratory Press; 1997. p. 131.1-131.27.10. Mayhew TM, Griffiths G, Lucocq JM. Applications of an efficient method for comparing immunogold labelling patterns in the same sets of compart-ments in different groups of cells. Histochem Cell Biol 2004; 122: 171-7.Zavašnik T et al. / Cystatin C in human dendritic cells 187Radiol Oncol 2006; 40(3): 183-8.11. Kos J, Štabuc B, Schweiger A, Krašovec M, Kopitar-Jerala M, Vrhovec I. Cathepsins B, H and L and their inhibitors stefin A and cystatin C in sera of melanoma patients. Clin Cancer Res 1997; 3: 1815-22.12. Kos J, Šmit A, Krašovec M, Svetic B, Lenarčič B, Vrhovec I, et al. Lysosomal proteases cathepsins D, B, H, L and their inhibitors stefins A and B in head and neck cancer. Biol Chem 1995; 376: 401-5.13. Cimerman N, Trstenjak-Prebanda M, Turk B, Popovič T, Dolenc I, Turk V. Interaction of cystatin C variants with papain and human cathepsins B, H and L. J Enzym Inhib 1999; 14: 167-74. 14. Kopitar G, Dolinar M, Štrukelj B, Pungerčar J, Turk V. Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli. Eur J Biochem 1996; 236: 558-62.15. Zavašnik-Bergant V, Sekirnik A, Golouh R, Turk V. Immunochemical localization of cathepsin S, cathepsin L and MHC II-associated p41 isoform of invariant chain in human lymph node tissue. Biol Chem 2001; 382: 799-804.16. Lennon-Duménil AM, Bakker AH, Maehr R, Fiebiger E, Overkleeft HS, Rosemblatt M, et al. Analysis of protease activity in live antigen-pre-senting cells shows regulation of the phagosomal proteolytic content during dendritic cell activa-tion. J Exp Med 2002; 196: 529-40.17. Zavašnik-Bergant T, Turk B. Cysteine cathepsins in the immune response. Tissue Antigens 2006; 67: 349-55.18. Pierre P, Turley SJ, Gatti E, Hull M, Meltzer J, Mirza A, et al. Developmental regulation of MHC II class II transport in mouse dendritic cells. Nature 1997; 388: 787-92.19. Pierre P, Mellman I. Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 1998; 93: 1135-45.Zavašnik T et al. / Cystatin C in human dendritic cells188Radiol Oncol 2006; 40(3): 183-8.Radiol Oncol 2006: 40(3): 175-81.Zaščita plodnosti pri bolnicah z rakomDel Pup L, Campagnutta E, Giorda G, De Piero G, Sopracordevole F, Sisto RIzhodišča. Biološko starševstvo je pomembno tudi za bolnike z rakom, zato vedno pogosteje že pred pričetkom onkološkega zdravljenja zaščitimo njihovo plodno sposobnost. Naloga onkologov je bolnike seznaniti z možnostjo, da bo onkološko zdravljenje lahko trajno okvarilo njihovo plodnost in kakšne so možnosti, da bi to jatrogeno okvaro zmanjšali ali preprečili. Metode zaščite plodnosti se zelo hitro razvijajo, čeprav o njih v strokovni litera-turi ne poročajo velikokrat. Da bi onkologom pomagali pri svetovanju bolnicam z rakom, v članku navajamo indikacije, kontraindikacije, omejitve in kontraverznosti različnih zaščit plodnosti.Zaključki. Ko onkolog obravnava bolnice z rakom, ki so v rodnem obdobju, naj bolnice seznani z možnimi posledicami zdravljenja in z načini, da bi bolnica ohranila plodno sposobnost. Priporočamo, da bolnico čim prej napoti na posvet k strokovnjaku, ki ima izkušnje z zdravljenjem plodnosti. Če se bolnice odločijo za eno od metod zaščite plodnosti, predlagamo vključitve v študijske raziskave, ki bodo lahko pokazale učinkovitost posamičnih metod.Radiol Oncol 2006; 40(3): 183-8.Različna lokalizacija cistatina C v nezrelih in zrelih dendritičnih celicahZavašnik-Bergant T, Bergant M, Jeras M, Griffiths GIzhodišča. Razgradnja antigenov s proteolitskimi encimi (proteazami) v endocitozni poti antigena predstavitvenih celic (dendritičnih celic) ter njihova regulacija z inhibitorji proteaz predstavlja pomemben korak pri nastanku antigenskih peptidov.Metode. Človeške dendritične celice so bile uporabljene kot celični model za študij pro-teaznega inhibitorja cistatina C. Pripravljene so bile tanke zamrznjene rezine nezrelih in zrelih dendritičnih celic ter označene s specifičnimi protitelesi za kvantitativno elektronsko mikroskopijo. Pod transmisijskim elektronskim mikroskopom so zrna koloidnega zlata, vezana na specifične sonde (protitelesa), pokazala natančno lokalizacijo označenega inhi-bitorja. Rezultati. Ovrednotenje označenih celic s statističnim testom Hi-kvadrat je potrdilo razlike v vsebnosti cistatina C v različnih celičnih organelih.Zaključki. Potrjena je bila statistično značilna razlika v znotrajcelični porazdelitvi cistatina C med populacijama nezrelih in zrelih dendritičnih celic.Slovenian abstracts 199Radiol Oncol 2006; 40(1): 197-200.

Different localisation of cystatin C in immature and mature dendritic cells

https://dirros.openscience.si/Dokument.php?id=24698&dn=

Different localisation of cystatin C in immature and mature dendritic cells

Abstract

Similar works

Full text

Available Versions

Digital repository of Slovenian research organizations