I. Current Research Interests
Among the conditions treated by rheumatologists are the systemic autoimmune diseases, with systemic lupus erythematosus (SLE) the prototype, systemic immune-mediated diseases that preferentially target the joints, with rheumatoid arthritis (RA) the prototype, and osteoarthritis, a degenerative joint disease. Cells and mediators of the immune system contribute to all of these disorders. The goal of the Immunoregulation Laboratory at Hospital for Special Surgery, directed by Dr. Peggy Crow, is to elucidate the underlying immune system mechanisms that account for development of autoimmunity and inflammation in these disorders, with the ultimate aim of identifying new approaches to more effectively treat these diseases. The major emphasis of the laboratory is on the mechanisms that trigger the initiation of autoimmunity and the development of altered immune function that results in cell-mediated tissue damage and autoantibody production. Current work uses SLE as a model disease for study. SLE patients recruited from the HSS patient population comprise the study subjects.
II. Mechanisms of initiation of SLE.
Immune responses can be divided into two phases: the innate immune response and the adaptive immune response. The innate immune response is triggered by components of microbes that interact with Toll-like receptors, pattern recognition receptors that activate the early phase of an immune response through the transcription factor NF-k B. The consequences of innate immune system activation include the activation of antigen-presenting cells, including dendritic cells, permitting more effective activation of the T cell- and B cell-mediated adaptive immune response, and production of cytokines that help to recruit inflammatory cells to the site of infection and to shape the functional pattern of the immune response.
The hypothesis being pursued by Dr. Crow's laboratory is that in parallel to the activation of the innate immune response by microbial organisms, endogenous triggers of an innate immune response can act as stimuli that promote initiation of autoimmunity and autoimmune disease. A current focus of the laboratory is the role of type I interferons, commonly induced in the setting of viral infection, in the initiation and pathogenesis of SLE.
The interferons (IFNs) were the first cytokines to be discovered more than 40 years ago. It is now known that there are subfamilies of IFNs, including type I IFNs -- IFNa, IFNß, IFNt, and IFNO -- and type II IFN -- IFN?, IFNß, and IFNa, which has 13 different isoforms, have similar sequences, a common receptor, and are made very early in response to viral infections; IFN?, on the other hand, is made later in the immune response, after antigen is presented to T cells.
Recent studies in Dr. Crow's laboratory have focused on the effects of IFNa. The laboratory was prompted to investigate the role of IFNa-induced gene expression in the pathogenesis of SLE because of the following data: (1) This cytokine is elevated in the serum of patients with active SLE; (2) IFNa induces intracellular vesicular inclusions in the renal endothelial cells of patients with SLE and lupus mice, suggesting that IFNa might have a functional impact on cells and possibly contribute to the pathogenesis of the disease; (3) Occasional patients who have received therapeutic administration of IFNa for viral infection or malignancy make typical lupus autoantibodies and in some cases develop clinical lupus; (4) Immune complexes containing lupus autoantibodies (autoantibody plus nucleic acid) and debris from apoptotic cells can induce the production of IFNa by plasmacytoid dendritic cells, the major immune system cells that produce IFNa; and (5) IFNa is one of several maturation factors for immature dendritic cells, turning them into more effective antigen-presenting cells.
Using sophisticated microarray analysis, Dr. Crow's laboratory worked with collaborators to show that IFN-induced genes are over-expressed in the peripheral blood of patients with SLE. Microarray analysis is used as a screen to identify genes expressed in a cell population at a given point in time. Dr. Crow's laboratory has used an array containing eight thousand oligonucleotide sequences derived from cDNA libraries isolated from resting and activated leukocytes which are spotted on a glass slide. RNA-derived material from the peripheral blood of patients with SLE is then hybridized to the gene array. Control RNA from patients with osteoarthritis, rheumatoid arthritis (RA), and juvenile chronic arthritis (JCA) were similarly hybridized to the gene array. The results indicate that, in contrast to the mRNAs from RA, JCA, and osteoarthritis patients and healthy controls, mRNAs encoded by IFN-regulated genes are among the most prominent observed in the peripheral blood of lupus patients. Similar results have been reported by several other groups, supporting the significance of the IFN pathway in SLE.
While some of the genes over-expressed in SLE are induced by both IFNa and IFN?, the patterns observed appear most consistent with a predominant IFNa, or other type I IFN, stimulus. Studies in Dr. Crow's laboratory, led by Dr. Kyriakos Kirou, using real-time polymerase chain reaction (PCR) analysis of panels of genes exclusively induced by either IFNa or IFN? support the conclusion that type I IFNs are primarily involved in gene induction in the peripheral blood of patients with SLE. PCR analysis has also confirmed the increased expression of IFNa genes in the blood of SLE patients with relatively active disease. Forty-five percent of the lupus population studied by the laboratory has an activated IFNa pathway, a figure which is likely to be partially dependent upon how active the disease is at the time blood is drawn. Activation of the IFNa pathway is not a characteristic of all patients with autoimmune or inflammatory diseases, since real-time PCR demonstrated that patients with RA have similar expression of IFN-induced genes as healthy controls. The laboratory continues to quantitatively analyze IFN-mediated regulation of gene expression in relation to clinical disease features. The laboratory has observed that a significant percentage of the population of patients with the activated IFNa pathway have autoantibodies to RNA-binding proteins. The investigators have been able to identify patients with more active disease and patients with a particular autoantibody profile by measuring the expression of the IFNa-induced genes. An important goal is to elucidate the pathophysiologic link between the ability to make IFNa and the expression of the class of autoantibodies that are specific for RNA-binding proteins.
The larger questions which Dr. Crow's laboratory seeks to answer by these types of experiments are how the IFNa pathway affects disease pathogenesis and what is the primary stimulus for IFN production. Dr. Crow postulates that IFNa might act as an "adjuvant"-like factor which could promote immune responses to self-antigens. IFNa provides an important link between the innate and adaptive immune systems. In the normal course of events, self-antigens, derived from apoptotic cells and other sources, may be expressed on the surface of antigen-presenting cells. When dendritic cells and other antigen-presenting cells are exposed to IFNa , they gain augmented capacity to stimulate autoantigen-reactive T cells and may trigger sustained activation of those self-reactive T cells. The T cells could then provide the "help" necessary for the production of autoantibodies by B cells. Therefore, the presence or absence of IFNa may contribute to the development of SLE in some individuals but not others.
The actions of IFNa on cells other than dendritic cells could also contribute to SLE pathogenesis. Not only does IFNa activate dendritic cells, but it enhances B-cell responses and promotes immunoglobulin class switching from IgM to IgG. IgG, unlike IgM, can enter the extravascular space and initiate inflammation in the skin and kidney. IFNa also promotes Fas ligand expression on natural killer cells, augmenting their capacity to mediate target cell apoptosis. Once IFNa is produced in large quantities, autoreactive T and B cell production may be enhanced, while at the same time the functional lymphocyte pool is depleted by apoptosis, contributing to an impaired capacity for an effective immune response to microbial attack.
While several potential mechanisms can be cited by which IFNa might induce the alterations in immune function that characterize SLE, the issue of why IFNa is produced is a more challenging question. Induction of the type I IFNs can be initiated through cell-surface Toll-like receptors as well as through intracellular signals induced by double stranded RNA. Some Toll-like receptors have specificity for lipopolysaccharide (LPS) found on E. coli; DNA enriched in demethylated cytosine and guanine, found in bacterial and viral DNA; and double-stranded or single-stranded RNA, found in viruses. While these receptors typically recognize various microbial products, they also have the potential to be activated by endogenous stimuli, such as human CpG DNA or RNA. DNA fragments generated during apoptosis or small RNAs encoded by genomic repeat elements or microRNAs are candidates for endogenous triggers of Toll-like receptor activation that might be relevant to SLE susceptibility. Some individuals may be particularly responsive to these endogenous triggers of Toll-like receptors and constitutively produce low levels of type I IFNs. Expression of genes regulated by type I IFNs might occur in a second phase of SLE, when low levels of autoantibodies combine with nucleic acids in the form of immune complexes and efficiently deliver the nucleic acid stimulus to intracellular Toll-like receptors. Increased levels of type I IFNs and induction of IFN-induced genes would follow and disease might then become clinically apparent. Some individuals may respond to this type I IFN production with the expression of IFN? and other pro-inflammatory gene products and represent those patients who go on to have significant inflammation and tissue damage in such organs as the skin and kidney. This model of disease is under investigation through the detailed analysis of gene expression and clinical features in a cohort of SLE patients followed at Hospital for Special Surgery.
The diverse data and observations indicating the importance of IFNa to the pathogenesis of SLE suggest that therapeutic targeting of the IFNa pathway may be a promising approach for treatment of SLE. The challenge will be to selectively inhibit the parts of the type I IFN pathway that contribute to disease while leaving intact sufficient IFNs and their receptors to maintain the efficacy of the immune response to microbial infection. There are thirteen known isoforms of IFNa, and currently little is known about how they interact or the conditions under which one or another IFN is expressed. Studies in Dr. Crow's laboratory are underway to determine whether some forms are produced preferentially in specific viral infections and others in SLE and whether they interact with their receptors in a similar manner. Additional studies are needed to understand which forms are involved in the pathogenesis of SLE. If this were known, then it might be possible to direct a therapeutic blockade to those specific IFNs involved in SLE while leaving intact those which provide a defense to microbial infection. It may be possible in the future to develop monoclonal antibodies that specifically block that IFNa pathway which is over-expressed in patients with SLE.
III. Mechanisms of tissue damage in SLE
A case control study directed by Dr. Jane Salmon at Hospital for Special Surgery and Dr. Mary Roman at Weill Medical College of Cornell University demonstrated that about 40% of patients with SLE develop atherosclerosis earlier than normal individuals, as measured by the presence of carotid artery plaques. Dr. Crow's laboratory is studying some of the mechanisms that may account for this prevalence of premature atherosclerosis. Experiments are underway to determine what cytokines and cytokine-induced gene products are expressed in these patients, with the goal of understanding how and why these SLE patients develop this additional complication. The real-time PCR technique is being used to quantify expression of pro-inflammatory cytokines and mediators that promote or control development of vascular lesions.
Dr. Crow has hypothesized that the patients with premature atherosclerosis are a distinct subgroup from those with activation of the IFNa pathway. Patients with atherosclerosis may have a more smoldering disease, where the inflammation builds up over time, compared to those who have high level IFN-induced genes and anti-RNA binding protein autoantibodies. Identification of some of the immune system characteristics of the group of SLE patients with atherosclerosis might permit screening of patients for those characteristics and detection of those patients at risk for this clinical manifestation at an earlier stage in their disease.
IV. Inflammation in osteoarthritis
While osteoarthritis is not usually considered an immune-mediated disease, Dr. Crow's laboratory has determined that approximately 50% of patients with osteoarthritis demonstrate infiltration of their synovial membrane with inflammatory cells. The laboratory is characterizing how certain cytokines and proteins interact in patients with osteoarthritis to generate joint inflammation and is determining the clinical consequences of that phenotype. The investigators have found that patients who have a high level of serum C-reactive protein, a marker of inflammation, and IL-6 in their joint fluid, are the patients with inflammatory infiltrates in their synovial membranes. The laboratory is currently investigating the gene expression patterns in the synovial tissues of patients with osteoarthritis, with the ultimate goal of understanding how inflammation contributes to disease progression.
Reviewed and Updated: 8/5/2009