David S. Pisetsky

Toll-like receptors in the pathogenesis of human disease.

Members of the Toll-like receptor (TLR) family are key regulators of both innate and adaptive immune responses. The function of TLRs in various human diseases has been investigated by comparison of the incidence of disease among people having different polymorphisms in genes that participate in TLR signaling. These studies have shown that TLR function affects several diseases, including sepsis, immunodeficiencies, atherosclerosis and asthma. As this body of data grows, it will provide new insights into disease pathogenesis as well as valuable information on the merits of various therapeutic options.

A Genomewide Screen in Multiplex Rheumatoid Arthritis Families Suggests Genetic Overlap with Other Autoimmune Diseases

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disorder with a complex genetic component. We report the first major genomewide screen of multiplex families with RA gathered in the United States. The North American Rheumatoid Arthritis Consortium, using well-defined clinical criteria, has collected 257 families containing 301 affected sibling pairs with RA. A genome screen for allele sharing was performed, using 379 microsatellite markers. A nonparametric analysis using SIBPAL confirmed linkage of the HLA locus to RA (P < .00005), with lambdaHLA = 1.79. However, the analysis also revealed a number of non-HLA loci on chromosomes 1 (D1S235), 4 (D4S1647), 12 (D12S373), 16 (D16S403), and 17 (D17S1301), with evidence for linkage at a significance level of P<.005. Analysis of X-linked markers using the MLOD method from ASPEX also suggests linkage to the telomeric marker DXS6807. Stratifying the families into white or seropositive subgroups revealed some additional markers that showed improvement in significance over the full data set. Several of the regions that showed evidence for nominal significance (P < .05) in our data set had previously been implicated in RA (D16S516 and D17S1301) or in other diseases of an autoimmune nature, including systemic lupus erythematosus (D1S235), inflammatory bowel disease (D4S1647, D5S1462, and D16S516), multiple sclerosis (D12S1052), and ankylosing spondylitis (D16S516). Therefore, genes in the HLA complex play a major role in RA susceptibility, but several other regions also contribute significantly to overall genetic risk.

Activation of human B cells by phosphorothioate oligodeoxynucleotides.

To investigate the potential of DNA to elicit immune responses in man, we examined the capacity of a variety of oligodeoxynucleotides (ODNs) to stimulate highly purified T cell-depleted human peripheral blood B cells. Among 47 ODNs of various sequences tested, 12 phosphorothioate oligodeoxynucleotides (sODNs) induced marked B cell proliferation and Ig production. IL-2 augmented both proliferation and production of IgM, IgG, and IgA, as well as IgM anti-DNA antibodies, but was not necessary for B cell stimulation. Similarly, T cells enhanced stimulation, but were not necessary for B cell activation. After stimulation with the active sODNs, more than 95% of B cells expressed CD25 and CD86. In addition, B cells stimulated with sODNs expressed all six of the major immunoglobulin VH gene families. These results indicate that the human B cell response to sODN is polyclonal. Active sODN coupled to Sepharose beads stimulated B cells as effectively as the free sODN, suggesting that stimulation resulted from engagement of surface receptors. These data indicate that sODNs can directly induce polyclonal activation of human B cells in a T cell-independent manner by engaging as yet unknown B cell surface receptors.

HMGB1: A multifunctional alarmin driving autoimmune and inflammatory disease

HMGB1 is a non-histone nuclear protein that can serve as an alarmin to drive the pathogenesis of inflammatory and autoimmune disease. Although primarily located in the cell nucleus, HMGB1 can translocate to the cytoplasm, as well as the extracellular space, during cell activation and cell death; during activation, HMGB1 can undergo post-translational modifications. The activity of HMGB1 varies with the redox states of the cysteine residues, which are required for binding to TLR4. In addition to stimulating cells directly, HMGB1 can form immunostimulatory complexes with cytokines and other endogenous and exogenous factors. In the synovia of patients with rheumatoid arthritis, as well as animal models of this disease, extranuclear expression of HMGB1 is increased and blockade of HMGB1 expression attenuates disease in animal models. In systemic lupus erythematosus, HMGB1 can be a component of immune complexes containing anti-DNA because of its interaction with DNA. In myositis, expression of HMGB1 is enhanced in inflamed muscle and can perturb muscle function. Together, these findings indicate that HMGB1 might be an important mediator and biomarker in rheumatic diseases as well as a target of new therapy.

Immune Activation by Bacterial DNA: A New Genetic Code

DNA is a molecule whose structural microheterogeneity encodes enormous information. Most of the information is complex as it conveys instructions for the precise structure and regulation of thousands of genes. Some of this information is simple, however, and provides in a direct and binary code data for the immune system to make the self–nonself discrimination. The extent to which the immune system uses this information is at present unknown, but the coming years should be intriguing as evidence is gathered to assess this unexpected potential of DNA.

The role of microparticles in the pathogenesis of rheumatic diseases

Microparticles (MPs) are small membrane-bound vesicles that are emerging as important elements in the pathogenesis of rheumatic diseases owing to their pleiotropic effects on thrombosis, vascular reactivity, angiogenesis and inflammation. Released from cells during activation and apoptosis, MPs carry proteins, lipids and nucleic acids, and serve as platforms for enzymatic processes in thrombosis. Furthermore, MPs can transfer cytokines, receptors, RNA and DNA to modulate the properties of target cells. As MPs appear in the blood in increased numbers during rheumatic disease, they represent novel biomarkers that can be used to assess events in otherwise inaccessible tissues. Future research will define further the pathogenetic role of MPs and explore therapeutic strategies to block their release or signaling properties.

Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA.

To investigate the role of antigen drive in anti-double-stranded (ds) DNA production, the antibody response induced in lupus-prone NZB/NZW mice by E. coli (EC) dsDNA was evaluated. Preautoimmune NZB/NZW female mice were immunized with complexes of EC dsDNA with methylated bovine serum albumin (mBSA) in complete Freunds adjuvant; control mice received either mBSA complexes with calf thymus (CT) dsDNA or mBSA alone in adjuvant. IgG antibody responses were assessed by ELISA. Similar to normal mice, immunized NZB/NZW mice produced significant levels of anti-dsDNA when measured with EC dsDNA as antigen. Whereas normal mice produce antibodies which are specific for the immunizing bacterial DNA, NZB/NZW mice produced antibodies that bound crossreactively to CT dsDNA by ELISA. Furthermore, the induced antibodies resembled lupus anti-DNA in their fine specificity for polynucleotide antigens and reactivity with Crithidia luciliae DNA. Despite their response to EC dsDNA, NZB/NZW mice immunized with CT dsDNA failed to generate significant anti-dsDNA responses. These results provide further evidence for the enhanced immunogenicity of bacterial DNA and suggest that immune cell abnormalities in NZB/NZW mice promote the generation of crossreactive autoantibody responses when confronted with a foreign DNA.

Interleukin‐1, tumor necrosis factor α, and interleukin‐17 synergistically up‐regulate nitric oxide and prostaglandin E2 production in explants of human osteoarthritic knee menisci

OBJECTIVE In osteoarthritis (OA), a combination of biochemical and biomechanical factors may damage both menisci and articular cartilage. Nitric oxide (NO) and prostaglandin E2 (PGE2) have been implicated as mediators of inflammation in OA. The goals of this study were to determine if menisci from patients with OA produce NO and PGE2, and if the proinflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor a (TNFalpha), and IL-17 augment NO and PGE2 production by these tissues. METHODS Menisci were obtained from 17 patients (age 47-75 years) undergoing total knee replacement for OA. Tissue explants were cultured alone or with IL-1beta, IL-17, or TNFalpha, and the release of NO and PGE2 from the tissue as well as the presence of type 2 nitric oxide synthase (NOS2) and cyclooxygenase 2 (COX-2) antigens were measured. RESULTS All menisci constitutively produced NO, and significant increases in NO production were observed in the presence of IL-1beta, TNFalpha, or IL-17 (P < 0.05). The combination of IL-17 and TNFalpha significantly increased NO production compared with either cytokine alone. Basal and cytokine-stimulated NO synthesis was inhibited by the NOS inhibitors NG-monomethyl-L-arginine or N-3-aminoethylbenzylacetamidine (1400W). IL-1beta significantly increased PGE2 production. The combination of IL-1beta and TNFalpha had an additive effect on PGE2 production, while addition of IL-17 to TNFalpha or IL-1beta synergistically enhanced PGE2 production. Inhibition of NO production by 1400W significantly increased IL-1beta-stimulated PGE2 production, and inhibition of PGE2 production by the COX-2 inhibitor N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide significantly increased IL-17-stimulated NO production. CONCLUSION Menisci from humans with OA spontaneously produced NO and PGE2 in a manner that was synergistically or additively augmented by cytokines. NO and PGE2 exhibited reciprocal regulatory effects on one another, suggesting that pharmaceutical agents designed to inhibit NOS2 or COX-2 production may in fact be influencing both pathways.

The Role of Microparticles in Inflammation and Thrombosis

Microparticles (MP) are small membrane‐bound vesicles that circulate in the peripheral blood and play active roles in thrombosis, inflammation and vascular reactivity. While MP can be released from nearly every cell type, most investigation has focused on MP of platelet, leucocyte and endothelial cell origin. Cells can release MP during activation or death. Flow cytometry is the usual method to quantify MP; the small size of these structures and lack of standardization in methodology complicate measurement. As MP contain surface and cytoplasmic contents of the parent cells and bear phosphatidylserine, antibodies to specific cell surface markers and annexin V can be used for identification. Through various mechanisms, MP participate in haemostasis and have procoagulant potential in disease. MP contribute to inflammation via their influence on cell–cell interactions and cytokine release, and MP also function in mediating vascular tone. In several disease states characterized by inflammation and vascular dysfunction, MP subpopulations are elevated, correlate with clinical events, and may have important roles in pathogenesis. In the rheumatic conditions such as rheumatoid arthritis and systemic lupus erythematosus, MP are potentially important markers of disease activity and have an increasingly recognized role in immunopathogenesis. It is clear that MP play an important role in atherosclerosis, and study of these structures may provide insight into the link between chronic inflammatory conditions and accelerated atherosclerosis. As biomarkers, MP allow access to usually inaccessible tissues such as the endothelium. Further research will hopefully lead to interventions targeting MP release and function.

High-mobility group box protein 1 (HMGB1): an alarmin mediating the pathogenesis of rheumatic disease

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that has a dual function. Inside the cell, HMGB1 binds DNA, regulating transcription and determining chromosomal architecture. Outside the cell, HMGB1 can serve as an alarmin to activate the innate system and mediate a wide range of physiological and pathological responses. To function as an alarmin, HMGB1 translocates from the nucleus of the cell to the extra-cellular milieu, a process that can take place with cell activation as well as cell death. HMGB1 can interact with receptors that include RAGE (receptor for advanced glycation endproducts) as well as Toll-like receptor-2 (TLR-2) and TLR-4 and function in a synergistic fashion with other proinflammatory mediators to induce responses. As shown in studies on patients as well as animal models, HMGB1 can play an important role in the pathogenesis of rheumatic disease, including rheumatoid arthritis, systemic lupus erythematosus, and polymyositis among others. New approaches to therapy for these diseases may involve strategies to inhibit HMGB1 release from cells, its interaction with receptors, and downstream signaling.