Expression of Cyclin B1 and Cyclin Dependent Kinase Inhibitor p21 in Lymphocytes in Patients with Systemic Lupus Erythematosus CHEONG-YIP HO, CHUN-KWOK WONG, EDMUND KWOK-MING LI, LAI-SHAN TAM, and CHRISTOPHER WAI-KEI LAM ABSTRACT. Objective. The roles of cyclins and cyclin dependent kinase (CDK) inhibitors of lymphocytes in the pathogenesis of systemic lupus erythematosus (SLE) are unclear. We measured the expression of cyclin B1 and CDK inhibitor p21 in peripheral blood lymphocytes (PBL) from patients with SLE and controls. Methods. PBL from 40 SLE patients with renal disease (RSLE), 40 SLE patients without renal disease (SLE), and 28 healthy control subjects were cultured with phytohemagglutinin (PHA). Bivariate distribution of cyclin B1 or p21 expression versus cellular DNA content was assessed by flow cytometry. Results. Expression of p21 in lymphocytes was significantly lower in patients with RSLE and with SLE than controls (RSLE vs controls and SLE vs controls, both p < 0.001). Expression of cyclin B1 was similar in all groups. The percentages of RSLE lymphocytes in G0/G1 and S phase were signif- icantly reduced and elevated, respectively, compared with controls. Conclusion. Downregulated p21 in PHA stimulated PBL from patients with SLE may be closely related to aberration of cell division in SLE lymphocytes. (J Rheumatol 2002;29:2537–44) Key Indexing Terms: SYSTEMIC LUPUS ERYTHEMATOSUS CYCLIN B1 CYCLIN DEPENDENT KINASE INHIBITOR p21 LYMPHOCYTES Systemic lupus erythematosus (SLE) is a complex autoim- tive T cells of MRL-lpr/lpr mice suggested that defective mune disease characterized by various immunological apoptosis might lead to increased cell cycling and hence abnormalities, including polyclonal activation of circulating disrupted tolerance toward self-antigens6,7. Another group B lymphocytes that produce a large quantity of autoreactive has reported that activated T cells from patients with SLE antibodies1,2. It is suggested that B cell proliferation in SLE differ in their cell cycle distribution8. Loss of tolerance and is T cell dependent, and the persistence of autoreactive B autoimmunity might thus stem from defects in cell division and T lymphocytes is thought to be responsible for the and dysregulated expression of cell cycle regulators. production of autoantibodies in SLE3. Following antigen Following mitogenic stimulation, quiescent cells (G0 exposure, mature lymphocytes require intense and repeated state) progress through 4 cell cycle phases: G1, the first gap proliferation to generate a rapid immune response and phase; S, DNA synthesis; G2, the second gap phase; and M, immunological memory. It has been established that dysreg- mitosis. Control of this progress involves positive regulators ulation of apoptosis in lymphocytes might lead to autoim- such as cyclins and cyclin dependent kinases (CDK), and munity, as illustrated by studies on lupus prone mice and negative regulators like CDK inhibitors9,10. During G1/S patients with SLE4,5. In vivo overproliferation of autoreac- phase progression, D cyclins (D1–D3) act in mid-G1, followed by cyclin E and cyclin A involved at the G1/S From the Department of Chemical Pathology and Department of boundary; cyclins A and B (B1 and B2) act during the S and Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong. G2/M phases. CDK require complexing with cyclins as well Supported by a Direct Grant for Research of The Chinese University of as phosphorylation for activity. CDK inhibitors function by Hong Kong and a donation from Zindart (De Zhen) Foundation Ltd., repressing the activity of cyclin-CDK complexes11. Based Hong Kong. on their structural characteristics and CDK targets, 2 classes C.Y. Ho, PhD, Postdoctoral Fellow; C.K. Wong, PhD, Associate of CDK inhibitors have been defined, Ink4 (inhibitors of Professor, Department of Chemical Pathology; E.K. Li, MD, Associate Professor; L.S. Tam, MD, Medical Officer, Department of Medicine and CDK4) and Cip/Kip (CDK interacting protein). Ink4 Therapeutics; C.W.K. Lam, PhD, Professor, Department of Chemical proteins including p15, p16, p18, and p19 associate solely Pathology. with CDK4 and CDK6, whereas the Cip/Kip proteins (p21, Address reprint requests to Prof. C.W.K. Lam, Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales p27, and p57) interfere with cycling by binding to Hospital, Shatin, NT, Hong Kong. E-mail: [email protected] cyclin/CDK complexes and inhibiting all CDK involved in Submitted January 14, 2002; revision accepted June 11, 2002. G1/S transition12,13. Personal non-commercial use only. The Journal of Rheumatology Copyright © 2002. All rights reserved. Ho, et al: Cell cycle in SLE 2537 Downloaded on September 30, 2021 from www.jrheum.org It is thought that p21 mediates cell cycle arrest by Wales Hospital, Hong Kong. Diagnosis of SLE was established according forming a quaternary complex with cyclins and their to the 1982 revised American Rheumatism Association criteria27, and complementary CDK, and thereby inhibiting the kinase disease activity was evaluated by the SLE Disease Activity Index (SLEDAI)28. The patients were divided in 2 groups: 40 SLE patients with 14 activity . It also binds to proliferating-cell nuclear antigen (or having the history of) renal disease (RSLE group) and 40 SLE patients (PCNA) for inhibiting DNA replication10. Malfunction of without renal disease (SLE group). Twenty-eight sex and age matched the p21 protein may contribute to the pathogenesis of healthy Chinese volunteers were recruited as controls. Twenty milliliters of systemic autoimmunity, e.g., SLE, through a variety of heparinized venous peripheral blood were collected from each patient and mechanisms. Inactivating mutations of p21 may allow over- control. The protocol of this study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong and informed activation and proliferation of otherwise quiescent, low consent was obtained from all participants. avidity, self-reactive T cells in the peripheral lymphoid Cell preparation. Peripheral blood lymphocytes (PBL) were isolated from organs of healthy individuals15. Studies on a p21–/– mouse heparinized venous blood by Ficoll-Paque density gradient centrifugation model showed that enhanced p21 deficient T cell prolifera- (Amersham Pharmacia Biotech, Uppsala, Sweden). The cells were washed tion was evident only after prolonged mitogenic T cell stim- twice with phosphate buffered saline (PBS; Sigma Chemical Co., St. Louis, ulation, a circumstance akin to repeated presentation of MO, USA), cultured in RPMI 1640 (Gibco Laboratories, Gaithersburg, MD, USA) supplemented with 10% fetal calf serum (Gibco), 20 mM 11 self-antigens to T cells . In the absence of p21, prolonged HEPES (Gibco), and 10 µg/ml PHA (Calbiochem, San Diego, CA, USA) exposure to antigen might thus lead to a disruption of toler- 6 in a density of 10 cells/ml at 37°C, 5% CO2. After 48 h, the cells were ance towards self-antigens, and the resulting accumulation harvested for flow cytometric analyses of cyclin B1 and p21. of CD4+ memory T cells might represent the conversion of Immunocytochemistry. After 48 h incubation with PHA, the cells were autoreactive cells to the memory phenotype16,17. rinsed with ice cold PBS and fixed in 80% ethanol at –20°C. After several Cyclin B1 is a mitotic cyclin that complexes with CDK1 washings with PBS, the cells were permeabilized with 0.25% Triton X-100 cdc2 (Sigma) in PBS for 5 min on ice. They were then washed and incubated (p34 ) to form the mitosis-promoting factor required for overnight at 4°C with murine monoclonal antibody to human cyclin B1 cells to enter mitosis. The onset of cyclin B1 synthesis starts (FITC conjugated IgG1, clone GNS-1; Becton Dickinson Pharmingen, San in G2 phase, with its expression peaking during the G2/M Diego, CA, USA) and p21 (IgG1, clone SX118; BD Pharmingen). The anti- transition, and its degradation being completed at the transi- p21 Mab was diluted in PBS containing 1% bovine serum albumin (BSA) tion to anaphase of mitosis18. Several studies have to obtain 0.25 µg/106 cells and both types of Mab were added in 100 µl. For confirmed that cyclin B1 is a key cell cycle regulator of the p21 staining, the cells were washed and incubated with a FITC conjugated goat anti-mouse IgG antibody (Zymed Laboratories, South San Francisco, 19-21 G2/M checkpoint . Cyclin B2 is a membrane associated CA, USA) diluted 1:40 in PBS containing 1% BSA for 30 min at room B-type cyclin that can also bind and activate CDK1. A temperature. The isotypic control was prepared identically as described recent study has suggested that cyclin B1 may compensate above, except that an isotype-specific antibody was used instead of cyclin for the loss of cyclin B2 in mutant cyclin B2-null mice and B1 or p21 Mab (mouse IgG1 for p21 Mab, Sigma; FITC conjugated mouse implies that cyclin B1 is capable of targeting the CDK1 to IgG1 of clone MOPC-21 for cyclin B1 Mab, BD Pharmingen). After 22 immunofluorescence staining, the cells were washed with PBS and resus- the essential substrates of cyclin B2 . An abundant level of pended in 10 µg/ml propidium iodide (Sigma) and 0.1% RNase A cyclin B1 has been found in the thymus of lupus prone (Calbiochem) in PBS, and incubated at room temperature for 20 min prior MRL-lpr/lpr mice compared to other strains, and southern to flow cytometric measurement. blot analysis of cyclin B1 gene showed restriction fragment Flow cytometry. The immunofluorescence stained cells were analyzed with length polymorphism and hence multiple forms of cyclin B1 a Becton Dickinson FACSCaliburTM flow cytometer. The red (propidium related sequences in various murine genomes23. However, to iodide) and green (FITC) emissions from each cell were separated and our knowledge no study on the expression of cyclin B1 in quantitated using the built-in FACSCalibur optics.