Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are inflammatory and destructive autoimmune diseases that are associated with increased morbidity and mortality Although the etiology of these diseases are unknown, recent studies have indicated that dendritic cells are macrophages are central to the pathogenesis of RA and SLE. However, the factors that control the survival, activation and function of dendritic cells and macrophages are unknown. To this end, we adopted a genetic approach to examine the role that cell cycle regulators and apoptosis inhibitors or inducers play during dendritic cell and macrophage development and activation. Currently, we have 41 single and double “knockout” mice and we have been establishing conditional knockout mice (only in dendritic cells and/or macrophages). Over the past several years we have shown that; 1) The cell cycle inhibitor p21 is essential for the development of inflammatory monocytes and for establishment of experimental arthritis. 2) Mice deficient in the Bcl-2 pro-apoptotic gene Bim have increased numbers of circulating monocytes, and have leukocytes which are refractory towards intrinsic apoptotic stimuli, and display increased development of experimental arthritis. 3) Mice lacking the death receptor Fas or its down stream effector protein Bid have macrophages that secrete higher levels of pro-inflammatory cytokines and chemokines and show enhanced development of experimental arthritis. 4) Mice lacking Bim and Fas display increased dendritic cell and macrophage activation and lupus like disease. These studies have led to novel insights into the essential factors that are responsible for maintaining homeostasis of dendritic cells and macrophages with respect to the development of experimental inflammatory arthritis or lupus. Below are four central projects in my laboratory that focus on the novel role that cell cycle and apoptotic molecules play in dendritic cells and macrophages and their contribution to RA and SLE pathogenesis.
1) The cyclin dependent kinase inhibitor p21 is a suppressor of acute inflammation. The mechanisms by which inflammation is controlled in the context of rheumatoid arthritis (RA) remain to be fully elucidated. The synovial lining which is comprised of fibroblasts and macrophages becomes hyperplastic in RA. Previous studies have suggested that the expression of the cyclin dependent kinase (CDK) inhibitors such as p21 (WAF1/CIP1), which functions to arrest the cell cycle by preventing CDKs from phosphorylating retinoblastoma protein (Rb), is reduced and thus contributes to the increased numbers of fibroblasts. However, in the RA synovium macrophages also have reduced expression of p21 even though they are terminally differentiated and have withdrawn from the cell cycle. Thus, the CDKi-CDK-Rb axis may play a crucial role in the activation and function of macrophages. Our preliminary data are the first to document this novel role for the CDKi-CDK-Rb axis and suggest that the CDKi, p21 is a novel suppressor of inflammation in macrophages. Macrophages lacking p21 exhibit increased activation and function. Additionally, p21-/- mice show increased susceptibility to LPS-induced endotoxic shock and enhanced development of inflammatory arthritis, two mouse models that require macrophages. Further, a peptide corresponding to the CDK binding domain of p21 is sufficient to mimic full length p21 and result in inhibition of pro-inflammatory cytokine production in isolated macrophages and reduction of inflammatory arthritis in mice.
2) Mimetics to Bim as a potential therapy for RA. In RA the balance between anti- and pro-apoptotic members of the Bcl-2 family may be shifted towards survival. While we demonstrated that the anti-apoptotic proteins, Bcl-2 and Mcl-1 are increased in RA synovial tissue compared to controls, no study has examined the therapeutic potential of antagonizing Bcl-2 anti-apoptotic members in arthritis. The study of deficiencies in anti-apoptotic members of the Bcl-2 family is complicated by embryonic lethality or early post-natal death. In contrast, mice deficient in pro-apoptotic Bcl-2 members such as Bak, Bax, and Bim survive and reach adulthood. Since Bim is a critical activator of apoptosis through sequestering Bcl-2/Mcl-1 and/or by activating Bak and Bax, Bim is a potential target for treatment of RA. We demonstrated that mice lacking the apoptotic initiator Bim but not the downstream effectors Bak or Bax develop a more severe form of inflammatory arthritis. This exacerbated disease in Bim-/- mice is associated with decreased apoptosis, increased expression of pro-inflammatory molecules, and more macrophages in pannus. Further, Bim-/- macrophages display elevated levels of IL-6 and TNFa, enhanced expression of CD40 and CD69, and increased and sustained level of active p38 in response to stimulation with LPS. Based on these data, we hypothesize that the ratio of Bim to Bcl-2 and/or Mcl-1 serves as molecular rheostat that determines the extent of hyperplasia and activation of macrophages in the joint. We will use a pharmacological approach, a whole animal approach, and a cell-specific approach to identify how deficiency in Bim exacerbates inflammatory arthritis. These studies will potentially lead to novel therapeutic approaches to RA.
3) Fas functions to inhibit the inflammatory response in macrophages. In previous studies we have shown that Fas, a death receptor and its ligand, FasL are present on the cell surface of peripheral blood monocytes and on joint macrophages and there is an increased expression of Flip, an inhibitor of Fas signaling, in joint macrophages isolated from RA patients. We have also demonstrated that mice mutant for Fas (lpr) have a more severe form of the K/BxN serum transfer-induced arthritis, a model of the effector phase of inflammatory arthritis. This exacerbated disease in lpr mice is associated with increased levels of cytokines and chemokines in the joint and more macrophages throughout the joint. Further, peritoneal macrophages from lpr mice produced markedly higher levels of TNFa and IL-1b as compared to wt mice. To extrapolate the specific role for Fas-signaling in macrophages in arthritis development we generated mice that conditionally lack Fas in monocytes and macrophages (CreF4/80-Fasflox/flox). These mice also develop an overly aggressive and worse form of arthritis as compared to wild-type (wt) mice. These data are the first to suggest that the expression of Fas in monocytes and macrophages is critical in determining the severity of the effector phase of inflammatory arthritis. Based on these data we hypothesize that Fas signaling is required in monocytes and macrophages to limit the inflammatory response in arthritis. This may occur by enhancing the level of Fas-mediated apoptosis, which would lead to a decrease in numbers of macrophages and subsequently reduced levels of TNFa and IL-1b. To dissect the mechanism through which Fas limits the pro-arthritic response in monocytes and macrophages we will use mice that conditionally lack Fas, caspase 8, and/or Flip only in monocytes and macrophages. These data are essential to understand the role of Fas-signaling in arthritis and for development of novel therapeutic approaches to treat RA.
4) Bim and Fas are suppressors of SLE pathogenesis. Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology characterized by production of autoantibodies and extensive end-organ damage. While it is clear that T and B lymphocytes are critical to SLE pathogenesis, intrinsic differences in antigen presenting cells (APCs) such as dendritic cells and macrophages, may influence the T-and B cell phenotypes observed in patients with SLE. The factors that control the activation, function, and/or survival of dendritic cells and macrophages, however, are poorly understood. We were the first to show that CD33+ dendritic cells and monocytes from lupus patients show an altered ratio of anti- to pro-apoptotic genes from both mitochondrial and death receptor pathways (Hutcheson et al Immunity 2008). Further, we and others demonstrated that mice deficient in pro-apoptotic members from mitochondrial (Bim) and death receptor (Fas) apoptotic pathways (Bim-/-Faslpr/lpr) exhibit an aggressive SLE-like disease on a normally resistant background and result in early lethality (<5 months). Dendritic cells and macrophages from Bim-/-Faslpr/lpr mice display increased expression in markers of activation, and induce enhanced allogeneic T-cell responses. In addition, renal dysfunction correlates with an increased presence of antigen presenting cells surrounding the glomeruli of Bim-/-Faslpr/lpr kidneys. We will use both a cell-specific approach and pharmacological approach to identify how a deficiency in apoptotic pathways leads to the development of SLE-like disease. These studies will potentially lead to novel therapeutics that alter the molecular rheostat controlling the fate of these cells and subsequently ameliorate SLE.
Selected Publications:
Lee D. Albee, Bo Shi, and Harris Perlman. Aspartic protease and caspase 3/7 activation is central for macrophage apoptosis following infection with Escherichia coli. 2007. Journal of Leukocyte Biology. 81:229-37.
Joslyn K. Brunelle, Emelyn H. Shroff, Harris Perlman, Andreas Strasser, Carlos T. Moraes, Richard A. Flavell, Nika N. Danial, Brian Keith, Craig B. Thompson, and Navdeep Chandel. Loss of Mcl-1 protein and inhibition of electron transport chain together induce anoxic cell death. 2007. Molecular and Cellular Biology. 27:1222-35.
Jack Hutcheson and Harris Perlman. Loss of Bim results in abnormal accumulation of mature CD4-CD8-CD44-CD25- thymocytes. 2007. Immunobiology. 212:629-36.
John C. Scatizzi, Emily Bickel, Jack Hutcheson, G. Kenneth Haines III, and Harris Perlman. Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis. 2007. Arthritis Research & Therapy. 9:R49.
Jack Hutcheson, John C. Scatizzi, Akbar Siddiqui, G. Kenneth Haines III, Tianfu Wu, Laurie Davis, Chandra Mohan, and Harris Perlman. 2008. Combined deficiency of Bim and Fas results in the early onset of systemic autoimmunity. Immunity. 28:206-17. (Comment in Immunity and Nature Reviews of Immunology).
Octavia M. Peck-Palmer, Jacqueline Unsinger, Katherine C. Chang, Jacquelyn S. McDonough, Harris Perlman, Jonathan E. McDunn, Richard S. Hotchkiss. (2009). Modulation of the Bcl-2 family blocks sepsis-induced depletion of dendritic cells and macrophages. Shock. 31(4):359-66.
John C. Scatizzi *, Melissa Mavers *, Jack Hutcheson, Brittany Young, Bo Shi, Richard M. Pope, Eric Ruderman, Damien S.K. Samways, John A. Corbett, Terrance M. Egan, and Harris Perlman. 2009. The cyclin dependent kinase domain of p21 is a suppressor of IL-1beta-mediated inflammation in activated macrophages. European Journal of Immunology. 39(3):820-5 (* denotes equal contribution)
