Induction of the measles virus receptor SLAM (CD150) on monocytes

Hiroko Minagawa1, Kotaro Tanaka1, Nobuyuki Ono1, Hironobu Tatsuo1 and Yusuke Yanagi1

Department of Virology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan1

Author for correspondence: Hiroko Minagawa. Fax +81 92 642 6140. e-mail hmina{at}virology.med.kyushu-u.ac.jp


   Abstract
Top
Abstract
Main text
References
 
Wild-type strains of measles virus (MV) isolated in B95a cells use the signalling lymphocyte activation molecule (SLAM; also known as CD150) as a cellular receptor, whereas the Edmonston strain and its derivative vaccine strains can use both SLAM and the ubiquitously expressed CD46 as receptors. Among the major target cells for MV, lymphocytes and dendritic cells are known to express SLAM after activation, but monocytes have been reported to be SLAM-negative. In this study, SLAM expression on monocytes was examined under different conditions. When freshly isolated from the peripheral blood, monocytes did not express SLAM on the cell surface. However, monocytes became SLAM-positive after incubation with phytohaemagglutinin, bacterial lipopolysaccharide or MV. Anti-SLAM monoclonal antibodies efficiently blocked infection of activated monocytes with a wild-type strain of MV. These results indicate that SLAM is readily induced and acts as a monocyte receptor for MV.


   Main text
Top
Abstract
Main text
References
 
Measles virus (MV) is a member of the genus Morbillivirus of the family Paramyxoviridae and naturally infects humans and subhuman primates. Measles is one of the leading causes of childhood mortality, with roughly 1000000 measles-associated deaths occurring worldwide per annum (Centers for Disease Control and Prevention, 1999 ). Measles-related mortality has been attributed mostly to MV-induced immune suppression (Griffin & Bellini, 1996 ; Klagge & Schneider-Schaulies, 1999 ).

Virus–receptor interaction plays an important role in virus tropism and pathogenesis (Schneider-Schaulies, 2000 ). CD46 (also known as membrane cofactor protein) has been identified as a cellular receptor for vaccine strains of MV, such as the Edmonston strain (Dörig et al., 1993 ; Naniche et al., 1993 ). It is expressed on all nucleated human cells and, accordingly, the Edmonston strain can infect almost any human cell line. In contrast, wild-type MV strains that are commonly isolated in the Epstein–Barr virus (EBV)-transformed marmoset B lymphocyte line B95a or human B cell lines are usually unable to use CD46 as a receptor and, thus, fail to infect many cell lines that are susceptible to the Edmonston strain (Bartz et al., 1998 ; Hsu et al., 1998 ; Lecouturier et al., 1996 ; Tanaka et al., 1998 ; Tatsuo et al., 2000 a). Recently, we have shown that the signalling lymphocyte activation molecule (SLAM; also known as CD150), a cell surface glycoprotein recognized by the monoclonal antibodies (MAbs) IPO-3 and A12 (Cocks et al., 1995 ; Sidorenko & Clark, 1993 ), is a cellular receptor for both wild-type and vaccine strains of MV (Tatsuo et al., 2000 b).

Unlike CD46, the known distribution of human SLAM is rather limited; it was identified originally as a molecule expressed on activated B and T cells (Cocks et al., 1995 ; Sidorenko & Clark, 1993 ). The use of SLAM as a receptor seems to serve well for MV as the virus grows better in activated cells rather than in resting ones (Hyypiä et al., 1985 ; Joseph et al., 1975 ). SLAM is also expressed constitutively on immature thymocytes, memory T cells and a proportion of B cells. Monocytes, natural killer (NK) cells (Aversa et al., 1997 ; Cocks et al., 1995 ; Sidorenko & Clark, 1993 ) and the myelomonocytic cell lines THP-1, U-937 and HL-60 (Aversa et al., 1997 ; Tatsuo et al., 2000 b) have little or no expression of SLAM. Dendritic cells (DCs) collected directly from blood express SLAM following activation via CD40 (Polacino et al., 1996 ). A recent study also reported SLAM expression on monocyte-derived DCs (Ohgimoto et al., 2001 ).

Besides T and B lymphocytes, MV has been shown to infect monocytes (Esolen et al., 1993 ; Joseph et al., 1975 ; Salonen et al., 1988 ), macrophages (Helin et al., 1999 ; Kamahora & Nii, 1965 ) and DCs (Fugier-Vivier et al., 1997 ; Grosjean et al., 1997 ; Schnorr et al., 1997 ; Steineur et al., 1998 ). Since all these cells may be responsible for systemic virus spread and MV-induced immune suppression, it is important to know how MV infects these cells. Lymphocytes and DCs may express SLAM as described above, whereas monocytes have been reported to be SLAM-negative. In this study, we examined the expression of SLAM on the monocyte cell surface under different conditions and the effects of anti-SLAM antibodies on MV infection of monocytes.

Peripheral blood mononuclear cells (PBMCs) were obtained from the leukocyte-enriched buffy coat of human peripheral blood by density gradient centrifugation at 600 g for 30 min onto Histopaque-1077 (Sigma) and were washed with PBS. MV stocks of the B95a-isolated KA and Ichinose strains (Tanaka et al., 1998 ; Tatsuo et al., 2000a , b ) and the Edmonston strain were prepared as described previously (Tanaka et al., 1998 ). For virus growth, B95a cells (Kobune et al., 1990 ) were grown in RPMI 1640 supplemented with 10% heat-inactivated foetal bovine serum, 0·18% sodium bicarbonate, 10 mM HEPES and 50 µg/ml gentamicin. To prepare the control inoculum, B95a cells without infected MV were treated similarly. UV-inactivated MV (UV-MV) was prepared by irradiating the virus stock with a UV cross-linker (Stratagene) at 240 mJ (Minagawa & Yanagi, 2000 ).

PBMCs were stained either immediately after isolation or 24 h after stimulation with one of the following reagents added to the growth medium: 5 µg/ml phytohaemagglutinin (PHA) (PHA-P; Sigma), 10 µg/ml lipopolysaccharide (LPS) from Escherichia coli (O55:B5; Sigma), MV with or without UV inactivation at an m.o.i. of 1 TCID50 per cell or control mock-infected B95a inoculum. Cells were suspended in wash buffer (PBS containing 0·1% BSA and 0·1% sodium azide) and incubated at room temperature for 1 h with MAbs [clone RMO2 (CD14; Coulter), clone A12 (SLAM; Pharmingen) or isotype controls] conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Cells were washed twice in the wash buffer and analysed on a FACScan flow cytometer (Becton-Dickinson; CellQuest software).

When freshly isolated PBMCs were examined, CD14+ cells comprising mostly monocytes were negative for SLAM (Fig. 1B), as reported previously (Cocks et al., 1995 ; Sidorenko & Clark, 1993 ). While SLAM expression on CD14+ cells increased slightly after incubation in growth medium (Fig. 1C, F), the majority of CD14+ cells became strongly SLAM-positive following stimulation with PHA (Fig. 1D). Stimulation with LPS also increased the proportion of SLAM-positive cells in the CD14+ cell population (Fig. 1E). Inoculation with either infectious MV (Fig. 1H) or UV-MV (Fig. 1I) also induced SLAM expression on CD14+ cells. Control inoculum prepared from mock-infected B95a cells only slightly induced SLAM expression (Fig. 1G). Since MV infection does not alter significantly the amounts of EBV secreted in the supernatants of B95a cells (Kobune et al., 1990 ), MV itself, but not EBV, must be largely responsible for this induction of SLAM. Induction of SLAM on CD14+ cells was observed reproducibly among seven donors after stimulation with PHA, LPS or UV-MV, prepared from two wild-type strains (KA and Ichinose). These results indicate that although freshly isolated monocytes do not express SLAM, they readily become SLAM-positive after activation.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1. Expression of SLAM on CD14+ cells. Experiments were repeated with seven different individual samples and representative data from two individual samples are shown: one for (A–E) and another for (F–I). Gates were set for mononuclear cells (lymphocytes and monocytes) and data were collected from at least 10000 events per tube. PHA-stimulated PBMCs were stained with FITC-conjugated (abscissa) and PE-conjugated (ordinate) isotype control MAbs (A). Freshly isolated PBMCs (B) and PBMCs incubated for 24 h in medium alone (C, F) or containing PHA (D), LPS (E), control B95a inoculum (G), infectious MV (H) or UV-MV (I) were stained for CD14 (abscissa) and SLAM (ordinate). The Ichinose strain of MV was used for this experiment.

 
To confirm that MV can infect monocytes by using SLAM as a receptor, we examined if anti-SLAM MAbs interfere with wild-type MV infection of PHA-stimulated monocytes. PBMCs were overlaid onto a discontinuous density Percoll (Pharmacia) gradient (75, 50·5, 40 and 30%) and centrifuged at 1000 g for 20 min at 4 °C (Takamizawa et al., 1997 ). The fraction with the lightest density was stimulated for 4 h with 5 µg PHA and then enriched further for monocytes by depleting T, B and NK cells as follows: cells were first incubated with mouse MAbs specific for CD3 (T cell marker), CD19 (B cell marker) and CD56 (NK cell marker) and then incubated with anti-mouse IgG covalently bound to paramagnetic polystyrene beads (Dynabeads; Dynal). Magnet-unbound cells were collected as purified monocytes and assessed for purity by flow cytometry. The percentage of CD14+ cells in the resultant population was >90%, while contaminating T and B cells were both less than 1%. These purified monocytes were pre-incubated with 10 µg/ml anti-SLAM MAb (IPO-3; Kamiya Biochemical) (Sidorenko & Clark, 1993 ) or control mouse IgG for 1 h, as described previously (Tatsuo et al., 2000 b). Cells were then inoculated with either the KA or the Edmonston strain of MV at an m.o.i. of 2 TCID50 per cell. After 1 h of adsorption, cells were washed and incubated for 18 h in fresh growth medium. Following extensive washing, cells were evaluated for MV haemagglutinin (H) expression by flow cytometry, as described previously (Tatsuo et al., 2000 b).

A large proportion of monocytes pre-treated with control mouse IgG expressed MV H protein on the cell surface following inoculation with the wild-type KA strain of MV (Fig. 2A). Monocytes pre-treated with IPO-3 hardly expressed H protein after inoculation with the KA strain. On the other hand, IPO-3 did not affect H protein expression on monocytes after inoculation with the Edmonston strain (Fig. 2B). This was expected as the Edmonston strain can use CD46 as well as SLAM to infect cells (Tatsuo et al., 2000 b). The inability of IPO-3 to inhibit infection with the Edmonston strain in turn indicates that it inhibited infection of monocytes with the KA strain by blocking the virus–SLAM interaction but not by affecting the intracellular virus replication following entry.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. Effect of anti-SLAM MAbs on MV infection of monocytes. Purified PHA-stimulated monocytes were treated with anti-SLAM (IPO-3; dotted line) or control IgG (thick line) and then inoculated with either the KA (A) or the Edmonston strain (B) of MV. At 18 h after inoculation, cells were washed and stained with MAbs against MV H protein and FITC-conjugated anti-mouse IgG. Control IgG-treated and infected monocytes were also stained only with anti-mouse IgG (thin line).

 
In this report, we showed that monocytes, which were SLAM-negative upon isolation from the peripheral blood, readily became SLAM-positive following incubation of PBMCs with PHA, LPS, infectious MV or UV-MV. The stimulation protocols used in the present study were simple and short in the time-course, unlike those used for the generation of macrophages (Helin et al., 1999 ) or DCs (Ohgimoto et al., 2001 ) from monocytes, indicating that monocytes do not need to be highly differentiated to express SLAM. This suggests that monocytes that migrate to the sites of inflammation and/or microbial propagation in vivo may become SLAM-positive under the influence of various stimuli, thus becoming susceptible to MV. Our finding provides a solution for the paradox that monocytes are major target cells for MV (Esolen et al., 1993 ; Salonen et al., 1988 ), yet do not express the MV receptor SLAM (Cocks et al., 1995 ; Sidorenko & Clark, 1993 ).

Anti-SLAM MAbs were shown to block infection of activated monocytes with the wild-type KA strain, indicating that SLAM indeed acts as a receptor for MV on monocytes. Some clinical isolates exclusively grown in PBMCs have been reported to use CD46 as a receptor (Manchester et al., 2000a ). However, we and others have shown that even those strains that use CD46 as a receptor are also capable of using SLAM (Erlenhoefer et al., 2001 ; Hsu et al., 2001 ; Tatsuo et al., 2000 b). Furthermore, we have shown that most viruses on throat swabs from measles patients use SLAM but not CD46 as a receptor (Ono et al., 2001 ).

Thus, SLAM is present not only on activated T and B lymphocytes and DCs, but also on activated monocytes. The distribution of SLAM, broader than that reported previously, fits the known tropism of MV as well as the multifactorial nature of MV-induced immune suppression (Borrow & Oldstone, 1995 ) to which infection of DCs (Fugier-Vivier et al., 1997 ; Grosjean et al., 1997 ; Schnorr et al., 1997 ; Steineur et al., 1998 ), interaction between viral proteins and cellular molecules (Manchester et al., 2000b ; Marie et al., 2001 ; Schlender et al., 1996 ) and soluble factors such as IL-12 (Karp et al., 1996 ) may all contribute. Further studies on the distribution of SLAM on other targets of MV infection (e.g. epithelial cells, endothelial cells and the central nervous system) and the function of SLAM expressed on the cells of the immune system should deepen our understanding of the pathogenesis of MV.


   Acknowledgments
 
We thank Dr S. Inaba, Blood Transfusion Service of Kyushu University Hospital and Dr Y. Maeda, Fukuoka Red Cross Blood Center, for human buffy coat; Dr F. Kobune for wild-type strains of MV and B95a cells and Ms Y. Kawanami for secretarial assistance. This work was supported by grants from the Ministry of Education, Science and Culture of Japan and from the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan.


   References
Top
Abstract
Main text
References
 
Aversa, G., Chang, C. C., Carballido, J. M., Cocks, B. G. & de Vries, J. E. (1997). Engagement of the signaling lymphocytic activation molecule (SLAM) on activated T cells results in IL-2-independent, cyclosporin A-sensitive T cell proliferation and IFN-{gamma} production. Journal of Immunology 158, 4036-4044.[Abstract]

Bartz, R., Firsching, R., Rima, B., ter Meulen, V. & Schneider-Schaulies, J. (1998). Differential receptor usage by measles virus strains. Journal of General Virology 79, 1015-1025.[Abstract]

Borrow, P. & Oldstone, M. B. A. (1995). Measles virus–mononuclear cells interactions. Current Topics in Microbiology and Immunology 191, 85-100.[Medline]

Centers for Disease Control and Prevention (1999). Global measles control and regional elimination, 1998–1999. Morbidity and Mortality Weekly Report 48, 1124–1130.[Medline]

Cocks, B. G., Chang, C. C., Carballido, J. M., Yssel, H., de Vries, J. E. & Aversa, G. (1995). A novel receptor involved in T-cell activation. Nature 376, 260-263.[Medline]

Dörig, R. E., Marcil, A., Chopra, A. & Richardson, C. D. (1993). The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 75, 295-305.[Medline]

Erlenhoefer, C., Wurzer, W. J., Löffler, S., Schneider-Schaulies, S., ter Meulen, V. & Schneider-Schaulies, J. (2001). CD150 (SLAM) is a receptor for measles virus but is not involved in viral contact-mediated proliferation inhibition. Journal of Virology 75, 4499-4505.[Abstract/Free Full Text]

Esolen, L. M., Ward, B. J., Moench, T. R. & Griffin, D. E. (1993). Infection of monocytes during measles. Journal of Infectious Diseases 168, 47-52.[Medline]

Fugier-Vivier, I., Servet-Delprat, C., Rivailler, P., Rissoan, M. C., Liu, Y. J. & Rabourdin-Combe, C. (1997). Measles virus suppresses cell-mediated immunity by interfering with the survival and functions of dendritic and T cells. Journal of Experimental Medicine 186, 813-823.[Abstract/Free Full Text]

Griffin, D. E. & Bellini, W. J. (1996). Measles virus. In Fields Virology , pp. 1267-1312. Edited by B. N. Fields, D. M. Knipe, P. M. Howley, R. M. Chanock, J. L. Melnick, T. P. Monath, B. Roizman & S. E. Straus. Philadelphia:Lippincott–Raven.

Grosjean, I., Caux, C., Bella, C., Berger, I., Wild, F., Banchereau, J. & Kaiserlian, D. (1997). Measles virus infects human dendritic cells and blocks their allostimulatory properties for CD4+ T cells. Journal of Experimental Medicine 186, 801-812.[Abstract/Free Full Text]

Helin, E., Salmi, A. A., Vanharanta, R. & Vainionpää, R. (1999). Measles virus replication in cells of myelomonocytic lineage is dependent on cellular differentiation stage. Virology 253, 35-42.[Medline]

Hsu, E. C., Sarangi, F., Iorio, C., Sidhu, M. S., Udem, S. A., Dillehay, D. L., Xu, W., Rota, P. A., Bellini, W. J. & Richardson, C. D. (1998). A single amino acid change in the hemagglutinin protein of measles virus determines its ability to bind CD46 and reveals another receptor on marmoset B cells. Journal of Virology 72, 2905-2916.[Abstract/Free Full Text]

Hsu, E. C., Iorio, C., Sarangi, F., Khine, A. A. & Richardson, C. D. (2001). CDw150 (SLAM) is a receptor for a lymphotropic strain of measles virus and may account for the immunosuppressive properties of this virus. Virology 279, 9-21.[Medline]

Hyypiä, T., Korkiamäki, P. & Vainionpää, R. (1985). Replication of measles virus in human lymphocytes. Journal of Experimental Medicine 161, 1261-1271.[Abstract]

Joseph, B. S., Lampert, P. W. & Oldstone, M. B. (1975). Replication and persistence of measles virus in defined subpopulations of human leukocytes. Journal of Virology 16, 1638-1649.[Medline]

Kamahora, J. & Nii, S. (1965). Pathological and immunological studies of monkeys infected with measles virus. Archiv für die Gesamte Virusforschung 16, 161-167.

Karp, C. L., Wysocka, M., Wahl, L. M., Ahearn, J. M., Cuomo, P. J., Sherry, B., Trinchieri, G. & Griffin, D. E. (1996). Mechanism of suppression of cell-mediated immunity by measles virus. Science 273, 228-231.[Abstract]

Klagge, I. M. & Schneider-Schaulies, S. (1999). Virus interactions with dendritic cells. Journal of General Virology 80, 823-833.[Free Full Text]

Kobune, F., Sakata, H. & Sugiura, A. (1990). Marmoset lymphoblastoid cells as a sensitive host for isolation of measles virus. Journal of Virology 64, 700-705.[Medline]

Lecouturier, V., Fayolle, J., Caballero, M., Carabaña, J., Celma, M. L., Fernandez-Muñoz, R., Wild, T. F. & Buckland, R. (1996). Identification of two amino acids in the hemagglutinin glycoprotein of measles virus (MV) that govern hemadsorption, HeLa cell fusion, and CD46 downregulation: phenotypic markers that differentiate vaccine and wild-type MV strains. Journal of Virology 70, 4200-4204.[Abstract]

Manchester, M., Eto, D. S., Valsamakis, A., Liton, P. B., Fernandez-Muñoz, R., Rota, P. A., Bellini, W. J., Forthal, D. N. & Oldstone, M. B. (2000a). Clinical isolates of measles virus use CD46 as a cellular receptor. Journal of Virology 74, 3967-3974.[Abstract/Free Full Text]

Manchester, M., Naniche, D. & Stehle, T. (2000b). CD46 as a measles receptor: form follows function. Virology 274, 5-10.[Medline]

Marie, J. C., Kehren, J., Trescol-Biémont, M.-C., Evlashev, A., Valentin, H., Walzer, T., Tedone, R., Loveland, B., Nicolas, J.-F., Rabourdine-Combe, C. & Horvat, B. (2001). Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14, 69-79.[Medline]

Minagawa, H. & Yanagi, Y. (2000). Latent herpes simplex virus-1 infection in SCID mice transferred with immune CD4+ T cells: a new model for latency. Archives of Virology 145, 2259-2272.[Medline]

Naniche, D., Varior-Krishnan, G., Cervoni, F., Wild, T. F., Rossi, B., Rabourdin-Combe, C. & Gerlier, D. (1993). Human membrane cofactor protein (CD46) acts as a cellular receptor for measles virus. Journal of Virology 67, 6025-6032.[Abstract]

Ohgimoto, S., Ohgimoto, K., Niewiesk, S., Klagge, I. M., Pfeuffer, J., Johnston, I. C. D., Schneider-Schaulies, J., Weidmann, A., ter Meulen, V. & Schneider-Schaulies, S. (2001). The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro. Journal of General Virology 82, 1835-1844.[Abstract/Free Full Text]

Ono, N., Tatsuo, H., Hidaka, Y., Aoki, T., Minagawa, H. & Yanagi, Y. (2001). Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. Journal of Virology 75, 4399-4401.[Abstract/Free Full Text]

Polacino, P. S., Pinchuk, L. M., Sidorenko, S. P. & Clark, E. A. (1996). Immunodeficiency virus cDNA synthesis in resting T lymphocytes is regulated by T cell activation signals and dendritic cells. Journal of Medical Primatology 25, 201-209.[Medline]

Salonen, R., Ilonen, J. & Salmi, A. (1988). Measles virus infection of unstimulated blood mononuclear cells in vitro: antigen expression and virus production preferentially in monocytes. Clinical and Experimental Immunology 71, 224-228.[Medline]

Schlender, J., Schnorr, J. J., Spielhoffer, P., Cathomen, T., Cattaneo, R., Billeter, M. A., ter Meulen, V. & Schneider-Schaulies, S. (1996). Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro. Proceedings of the National Academy of Sciences, USA 93, 13194-13199.[Abstract/Free Full Text]

Schneider-Schaulies, J. (2000). Cellular receptors for viruses: links to tropism and pathogenesis. Journal of General Virology 81, 1413-1429.[Free Full Text]

Schnorr, J. J., Xanthakos, S., Keikavoussi, P., Kampgen, E., ter Meulen, V. & Schneider-Schaulies, S. (1997). Induction of maturation of human blood dendritic cell precursors by measles virus is associated with immunosuppression. Proceedings of the National Academy of Sciences, USA 94, 5326-5331.[Abstract/Free Full Text]

Sidorenko, S. P. & Clark, E. A. (1993). Characterization of a cell surface glycoprotein IPO-3, expressed on activated human B and T lymphocytes. Journal of Immunology 151, 4614-4624.[Abstract/Free Full Text]

Steineur, M. P., Grosjean, I., Bella, C. & Kaiserlian, D. (1998). Langerhans cells are susceptible to measles virus infection and actively suppress T cell proliferation. European Journal of Dermatology 8, 413-420.[Medline]

Takamizawa, M., Rivas, A., Fagnoni, F., Benike, C., Kosek, J., Hyakawa, H. & Engleman, E. G. (1997). Dendritic cells that process and present nominal antigens to naive T lymphocytes are derived from CD2+ precursors. Journal of Immunology 158, 2134-2142.[Abstract]

Tanaka, K., Xie, M. & Yanagi, Y. (1998). The hemagglutinin of recent measles virus isolates induces cell fusion in a marmoset cell line, but not in other CD46-positive human and monkey cell lines, when expressed together with the F protein. Archives of Virology 143, 213-225.[Medline]

Tatsuo, H., Okuma, K., Tanaka, K., Ono, N., Minagawa, H., Takade, A., Matsuura, Y. & Yanagi, Y. (2000a). Virus entry is a major determinant of cell tropism of Edmonston and wild-type strains of measles virus as revealed by vesicular stomatitis virus pseudotypes bearing their envelope proteins. Journal of Virology 74, 4139-4145.[Abstract/Free Full Text]

Tatsuo, H., Ono, N., Tanaka, K. & Yanagi, Y. (2000b). SLAM (CDw150) is a cellular receptor for measles virus. Nature 406, 893-897.[Medline]

Received 8 August 2001; accepted 30 August 2001.