Bovine Doppel (Dpl) and Prion Protein (PrP) Expression on Lymphoid Tissue and Circulating Leukocytes
Department of Veterinary Pathology, Hygiene and Health, University of Milan, Milan, Italy
Correspondence to: Prof. Saverio Paltrinieri, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università di Milano, Via Celoria 10, 20133 Milano, Italy. E-mail: saverio.paltrinieri{at}unimi.it
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(J Histochem Cytochem 52:16391645, 2004)
Key Words: doppel prion leukocytes follicular dendritic cells immunohistochemistry flow cytometry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Studies using Western blot and RT-PCR have shown that the tissue distribution of Dpl in both humans and bovine is similar to that reported in mice (Sakaguchi et al. 1996; Moore et al. 1999
; Tranulis et al. 2001
; Rondena et al. 2004
). Interestingly, bovine Dpl has also been visualized by immunohistochemistry on the membrane of scattered cells in the spleen and lymph nodes (Rondena et al. 2004
). Previous studies have shown that PrP is also expressed in follicular dendritic cells (FDCs) (Thielen et al. 2001b
) and in several types of blood cells in various species (Herrmann et al. 2001
; Barclay et al. 2002
). The expression of PrP in FDCs is essential for neuroinvasion (Brown et al. 1999
; Mabbot et al. 2000
), while the role of PrP-expressing leukocytes is still debated (Klein et al. 1998
; Li et al. 2001
).
Because of the above-mentioned similarities between PrP and Dpl and the possible role of Dpl in the pathogenesis of experimentally induced (PrP deletion) or spontaneous (infertility, brain tumor) diseases, it is important to understand more about Dpl biology. This article describes the results of a study focused on the expression of Dpl in both circulating and tissue leukocytes and on the relationship between Dpl and PrP expression. For these purposes, bovine cells and tissues were used owing to the relevance of cattle in prion-related diseases.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blood was collected by venipuncture from the coccygeal vein of six adult Holstein-Fresian cattle. Animals came from a dairy farm and were clinically healthy. Blood was collected in EDTA-coated tubes. A routine complete cell blood count using an automated analyzer (Hemat 8; SEAC, Firenze, Italy) followed by microscopic evaluation of differential leukocyte count on May-Grünwald-Giemsa-stained smears was performed to exclude the presence of subclinical hematological abnormalities. Blood was then used to perform flow cytometry.
Bovine semen was used as a positive control for flow cytometric detection of Dpl due to the established expression of the protein in spermatozoa (Rondena et al. 2004). Frozen bovine semen, routinely stored for bovine artificial insemination at the Department of Clinical Sciences of the Veterinary School of Milan, was used. The semen was thawed and washed with PBS to remove cryopreservatives. Spermatozoa were then counted, resuspended in RPMI supplemented with 5% fetal calf serum (FCS) and 0.2% sodium azide to a final dilution of 1 x 106 cells/ml, and analyzed by flow cytometry.
Immunohistochemistry
Immunohistochemistry was performed on cryostat sections using the avidin-biotin complex (ABC) technique using a commercially available kit (Vector Laboratories; Burlingame, CA). Endogenous peroxidases were inhibited with H2O2 (1%) in 0.1% sodium azide. The blocking solution was prepared by mixing 2% milk powder in Tris-buffered saline (TBS), pH 7.6, with normal goat (for the polyclonal antibody) or horse (for monoclonal antibodies) serum. The blocking solution was then applied (30 min at room temperature) to the slides. The primary antibodies listed in Table 1 were diluted in blocking solution and applied to the slides for 1 hr at 37C. The optimal working dilution for each antibody was established by preliminary tests with serial dilution. After three washes in TBS, the biotinylated secondary antibody (30 min at room temperature) and the ABC complex (30 min at room temperature) were added sequentially. Either diaminobenzidine tetrahydrochloride (DAB; Vector Laboratories) or 3-amino-9-ethyl-carbazole (AEC; Vector Laboratories) was used as chromogen for visualization. The reaction was blocked by rinsing in running tap water and the slides were counterstained with Mayer's hematoxylin and coverslipped using non-aqueous mounting medium (Eukitt, Kindler; Freiburg, Germany). Selected histological sections were used as negative controls using normal rabbit serum (DAKO; Glostrup Denmark) as the primary antibody. As positive controls, a section of ovary and a section of testis from adult bovine, for which the distribution of Dpl-positive cells has already been standardized (Rondena et al. 2004), was also included.
|
Flow Cytometry
Spermatozoa were incubated with the polyclonal anti-Dpl antibody or with a negative rabbit IgG (DAKO) as a control, for 20 min at 4C. They were washed twice, resuspended, and incubated with the secondary antibody (phycoerythrin-conjugated anti-rabbit IgG; Serotec, Oxford, UK) for 20 min at 4C. Blood samples (0.5 ml) were lysed in 7 ml of RBC lysing solution (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM tetrasodium EDTA in distilled water). After washing, leukocytes were diluted in RPMI containing 5% FCS and 0.2% sodium azide at a final concentration of 1 x 106 cells/ml. Cells were first incubated with the polyclonal anti-Dpl antibody at the dilution established in a previous work (Rondena et al. 2004) or with a negative rabbit IgG (DAKO) as a control, for 20 min at 4C. Cells were then incubated with the secondary antibody (phycoerythrin-conjugated anti-rabbit IgG) for 20 min at 4C. Double staining was performed in a second step, using monoclonal antibodies or isotype-matched murine control sera (Serotec). To establish the best working dilution of each monoclonal antibody, titration of each antibody was performed starting from the dilution suggested by the manufacturers. Negative controls were used at the same IgG concentration. After incubation and washing, the secondary antibody (fluorescein-conjugated rabbit anti-mouse IgG; Serotec) was added.
Both spermatozoa and leukocytes were resuspended in 0.5 ml PBS. Fluorescence was measured on a minimum of 10,000 cells using a FACSort flow cytometer (Becton Dickinson; San Jose, CA). Data were analyzed using CELLQuest software (Becton Dickinson).
Positivity for CD45 was used to distinguish leukocytes from debris and to backgate each population (Figure 1). The percentage of positive events for Dpl in each leukocyte subset or in PrP-expressing cells was then counted and compared with that recorded in control cell population by means of the Kolmogorov Smirnov test. Statistical significance was established at p<0.001.
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Flow Cytometry
The flow cytometric protocol to detect Dpl was preliminarily assessed on ejaculated spermatozoa, in which the expression of Dpl was already documented by flow cytometry in humans (Peoc'h et al. 2002) and bovine (Rondena et al. 2004
). Using ejaculated spermatozoa as a positive control, the expression of Dpl and its possible relationship with PrP expression was then investigated in blood cells. The results are summarized in Table 2 and shown in Figure 3, which clearly demonstrates the presence of Dpl-expressing cells in different leukocyte subpopulations. Specifically, a positive signal was detected in CD11b+, CD11c+ polymorphonuclear granulocytes (PMNs), and in CD21+ B-lymphocytes, while CD14+ monocytes and other lymphocyte subsets and platelets had weak to absent Dpl positivity (Table 2).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dpl and PrP appear to be expressed in different cell populations in blood. PrP in ruminants has been reported to be expressed in B-cells and, to a lesser extent, in other lymphocyte subsets and monocytes (Herrmann et al. 2001; Barclay et al. 2002
). PrP expression in platelets and PMNs has been reported to be low (Herrmann et al. 2001
) or absent (Barclay et al. 2002
). Our flow cytometric results basically confirm these findings with regard to PrP. Similarly to PrP, Dpl is not expressed on platelets and its expression in B-cells is higher than in other lymphocyte subsets. The strongest Dpl expression, however, was found in circulating PMNs. This finding is in agreement with the immunohistochemical detection of rare positive myeloid cells in bone marrow. This suggests that, contrary to what has been demonstrated for PrP (Dodelet and Cashman 1998
), the expression of Dpl increases during PMN maturation and Dpl appears to be downregulated during platelet maturation. Interestingly, previous immunohistochemical investigations did not detect Dpl in resident macrophages or scattered phagocytes that can be occasionally found in bovine tissues (Rondena et al. 2004
). Moreover, in the present study myeloid cells identified by antibodies directed against CD11b, CD11c, or CD14 did not stain for Dpl in lymphoid tissues. Resting and/or non-circulating phagocytes therefore appear to downregulate Dpl, suggesting that the activation state might influence Dpl expression, as already demonstrated for PrP, which is differentially expressed during maturation of leukocytes or platelets (Holada et al. 1998
; Durig et al. 2000
). This possibility is supported by our observation that many Dpl-expressing lymphocytes co-express MHCII. The possible expression of Dpl in activated cells merits further investigation, because it cannot be excluded that phagocytes recruited to inflamed sites might express Dpl.
It has been demonstrated that PrP and Dpl are expressed on different membrane rafts (Shaked et al. 2002), and the observed coexpression of Dpl and PrP in blood and tissues confirms this evidence. However, the number of cells expressing both proteins was very low, suggesting that the mechanisms involved in Dpl expression in resting blood cells are probably different from and independent of those responsible for PrP expression. Likewise, the possible coexpression of PrP and Dpl and the influence of PrP on Dpl expression (and vice versa) would be of interest to investigate in blood and tissues from animals affected by TSE and/or in an experimental model based on PrP/Dpl blocking or deletion.
Other factors that might influence the expression of Dpl in blood and lymphoid cells remain to be elucidated. On the basis of the results of previous reports (Rondena et al. 2004) that did not show any age-dependent differential expression of bovine Dpl, it might reasonably be excluded that age might influence Dpl expression in blood. However, the possible influence exerted by host genetic factors in conditioning different expression levels of both Dpl and PrP in bovine leukocytes must also be examined because prnd, the Dpl-encoding gene in bovine, has been shown to be polymorphic (Comincini et al. 2001
).
The information obtained from this study might therefore be translated to murine models of PrP-induced diseases or to the analysis of human PrP and Dpl biology because additional homologies between the two proteins have been identified. However, in spite of a similar distribution in both lymphoid cells and tissues, PrP and Dpl are rarely co-expressed, and the following differences in the expression pattern of the two proteins were detected: (a) the number of cells expressing Dpl is lower than that expressing PrP; (b) PMNs express Dpl but not PrP; and (c) Dpl expression appears to vary with the activation state of the cells. Our results further support the hypothesis that Dpl is not involved in spontaneous TSE. Nevertheless, blood might be easily used to design in vitro tests to differentially enhance or suppress the expression of the two proteins to gain useful information about the pathobiology of Dpl and PrP.
![]() |
Acknowledgments |
---|
We are very grateful to Prof Giorgio Cammarata, Prof Cecilia Luvoni, and Dr Sara Chigioni (University of Milan) and to Prof Luca Ferretti (University of Pavia).
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barclay GR, Houston EF, Halliday SI, Farquhar CF, Turner ML (2002) Comparative analysis of normal prion protein expression on human, rodent, and ruminant blood cells by using a panel of prion antibodies. Transfusion 42:517526[CrossRef][Medline]
Behrens A, Genoud N, Naumann H, Rulicke T, Janett F, Heppner FL, Ledermann B, et al. (2002) Absence of the prion protein homologue Doppel causes male sterility. EMBO J 21:36523658
Brown KL, Stewart K, Ritchie DL, Mabbot NA, Williams A, Fraser H, Morrison WI, et al. (1999) Scrapie replication in lymphoid tissues depends on prion protein-expressing follicular dendritic cells. Nat Med 5:13081312[CrossRef][Medline]
Comincini S, Facoetti A, Del Vecchio I, Peoc'h K, Laplanche JL, Magrassi L, Ceroni M, et al. (2004) Differential expression of the prion-like protein doppel gene (PRND) in astrocytomas: a new molecular marker potentially involved in tumor progression. Anticancer Res 24:15071517[Medline]
Comincini S, Foti MG, Tranulis MA, Hills D, Di Guardo G, Vaccari G, Williams JL, et al. (2001) Genomic organization, comparative analysis, and genetic polymorphisms of the bovine and ovine prion Doppel genes (PRND). Mamm Genome 12:729733[CrossRef][Medline]
Cui T, Holme A, Sassoon J, Brown DR (2003) Analysis of doppel protein toxicity. Mol Cell Neurosci 23:144155[CrossRef][Medline]
Dodelet VC, Cashman NR (1998) Prion protein expression in human leukocyte differentiation. Blood 91:15561561
Durig J, Giese A, Schultz-Schaeffer W, Rosenthal C, Schmucker U, Bieschke J, Duhrsen U, et al. (2000) Differential and constitutive and activation-dependent expression of prion protein in human peripheral blood leucocytes. Br J Haematol 108:488495[CrossRef][Medline]
Herrmann LM, Davis WC, Knowles DP, Wardrop KJ, Sy MS, Gambetti P, O' Rourke KI (2001) Cellular prion protein is expressed on peripheral blood mononuclear cells but not platelets of normal and scrapie-infected sheep. Haematologica 86:146153[Medline]
Holada K, Mondoro TH, Muller J, Vostal JG (1998) Increased expression of phosphatidylinositol-specific phospholipase C resistant prion proteins on the surface of activated platelets. Br J Haematol 103:276282[CrossRef][Medline]
Klein MA, Frigg R, Raeber AJ, Flechsig E, Hegyi I, Zinkernagel RM, Weissmann C, et al. (1998) PrP expression in B lymphocytes is not required for prion neuroinvasion. Nat Med 4:14291433[CrossRef][Medline]
Li A, Sakaguchi S, Shigematsu K, Atarashi R, Roy BC, Nakaoke R, Arima K, et al. (2002) Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration. Am J Pathol 157:14471452
Li R, Liu D, Zanusso G, Liu T, Fayen JD, Huang JH, Petersen RB, et al. (2001) The expression and potential function of cellular prion protein in human lymphocytes. Cell Immunol 207:4858
Luhrs T, Riek R, Guntert P, Wuthrich K (2003) NMR structure of the human doppel protein. J Mol Biol 326:15491557[CrossRef][Medline]
Mabbot NA, MacKay F, Minns F, Bruce ME (2000) Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie. Nat Med 6:719720[CrossRef][Medline]
Makrinou E, Collinge J, Antoniou M (2002) Genomic characterization of the human prion protein (PrP) gene locus. Mamm Genome 13:696703[CrossRef][Medline]
Moore RC, Lee IY, Silverman GL, Harrison PM, Strome R, Heinrich C, Karunaratne A, et al. (1999) Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel. J Mol Biol 292:797817[CrossRef][Medline]
Peoc'h K, Serres C, Frobert Y, Martin C, Lehmann S, Chasseigneaux S, Sazdovitch V, et al. (2002) The human "prion-like" protein Doppel is expressed in both Sertoli cells and spermatozoa. J Biol Chem 277:4307143078
Rondena M, Ceciliani F, Comazzi S, Pocacqua V, Bazzocchi C, Luvoni C, Chigioni S, et al. (2004) Identification of bovine doppel protein in testis, ovary and ejeculated spermatozoa. Theriogenology in press
Sakaguchi S, Katamine S, Nishida N, Moriuchi R, Shigematsu K, Sugimoto T, Nakatani A, et al. (1996) Loss of cerebellar Purkinje cells in aged mice homozygous for a disrupted PrP gene. Nature 380:528531[CrossRef][Medline]
Shaked Y, Hijazi N, Gabizon R (2002) Doppel and PrP(C) do not share the same membrane microenvironment. FEBS Lett 530:8588[CrossRef][Medline]
Thielen C, Antoine M, Mélot F, Cesbron J-Y, Heinen E, Tsunoda R (2001a) Human FDC express PrPc in vivo and in vitro. Dev Immunol 8:259266[Medline]
Thielen C, Mélot F, Jolois O, Leclercq F, Tsunoda R, Frobert Y, Heinen E, et al. (2001b) Isolation of bovine follicular dendritic cells allows the demonstration of a particular cellular prion protein. Cell Tissue Res 306:4955[CrossRef][Medline]
Tranulis MA, Espenes A, Comincini S, Skretting G, Harbitz I (2001) The PrP-like protein Doppel gene in sheep and cattle: cDNA sequence and expression. Mamm Genome 12:376379[CrossRef][Medline]