-
H L Rosenzweig,
M M Jann,
T T Glant,
T M Martin,
S R Planck,
W van Eden,
P J S van Kooten,
R A Flavell,
K S Kobayashi,
J T Rosenbaum, M P Davey
[show abstract]
[hide abstract]
ABSTRACT: In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2-deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR-transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen-specific activation of splenocytes was enhanced by MDP with respect to IFN-gamma production, which would be consistent with the Th1-mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG-induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.
Journal of leukocyte biology 02/2009; 85(4):711-8. · 4.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nucleotide-binding and oligomerization domain 2 (NOD2) belongs to the emerging Nod-like receptor (NLR) family considered important in innate immunity. Mutations in NOD2 cause Blau syndrome, an inherited inflammation of eye, joints, and skin. Mutations in a homologous region of another NLR member, NALP3, cause autoinflammation, wherein IL-1beta plays a critical role. Here, we tested the hypothesis that IL-1beta is a downstream mediator of NOD2-dependent ocular inflammation. We used a mouse model of NOD2-dependent ocular inflammation induced by muramyl dipeptide (MDP), the minimal bacterial motif sensed by NOD2. We report that MDP-induced ocular inflammation generates IL-1beta and IL-18 within the eye in a NOD2- and caspase-1-dependent manner. Surprisingly, two critical measures of ocular inflammation, leukocyte rolling and leukocyte intravascular adherence, appear to be completely independent of IL-1 signaling effects, as caspase-1 and IL-1R1-deficient mice still developed ocular inflammation in response to MDP. In contrast to the eye, a diminished neutrophil response was observed in an in vivo model of MDP-induced peritonitis in caspase-1-deficient mice, suggesting that IL-1beta is not essential in NOD2-dependent ocular inflammation, but it is involved, in part, in systemic inflammation triggered by NOD2 activation. This disparity may be influenced by IL-1R antagonist (IL-1Ra), as we observed differential IL-1Ra levels in the eye versus plasma at baseline levels and in response to MDP treatment. This report reveals a new in vivo function of NOD2 within the eye yet importantly, distinguishes NOD2-dependent from NALP3-dependent inflammation, as ocular inflammation in mice occurred independently of IL-1beta.
Journal of Leukocyte Biology 06/2008; 84(2):529-36. · 4.99 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Uveitis is often associated with a systemic inflammatory disease such as ankylosing spondylitis. Our understanding of the eye's susceptibility to immune-mediated uveitis as in the apparent absence of infection has been limited by a relative lack of experimental models. Here we sought to assess whether ocular inflammation occurs in a previously described murine model of proteoglycan-induced spondylitis, wherein mice develop progressive spondylitis, sacroiliitis and peripheral arthritis--features common to the clinical presentations of ankylosing spondylitis.
Using intravital microscopy we examined the ocular inflammatory response after the onset of arthritis in mice that overexpressed the T cell receptor (TCR) specific for a dominant arthritogenic epitope of cartilage proteoglycan [TCR-Tg (transgenic) mice] or BALB/c controls.
Immunized TCR-Tg mice showed a significant increase in the number of rolling and adhering cells within the iris vasculature compared to adjuvant control mice. Cellular infiltration within the iris tissue, as assessed by intravital microscopy and histology, was also increased. Our initial temporal analysis has revealed that immunized TCR-Tg mice show a significant increase in intravascular inflammation by 2 weeks after immunization, but it diminishes at 4 weeks after immunization.
Although these data are preliminary, this model has the potential to clarify the mechanisms accounting for the coexistence of eye and sacroiliac inflammation as occurs in patients with ankylosing spondylitis.
Ophthalmic Research 02/2008; 40(3-4):189-92. · 1.56 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mutations in the human NOD2/CARD15 gene have been associated with Crohn's disease and Blau syndrome. The objective of the present study was to clone the murine form of NOD2 and characterize its tissue distribution, function and response to inflammatory stimuli.
Murine NOD2 was isolated using anchored polymerize chain reaction (PCR). Sequence analysis confirmed the identification of full-length cDNA representing the murine NOD2 gene. Using this sequence to search a Mus musculus supercontig database, NOD2 genomic DNA was identified. NOD2 was transfected into human embryonic kidney (HEK) cells and nuclear factor kappa B (NF-kappaB) activation was measured using a reporter assay. Tissue distribution and changes in transcription in mouse monocytes in response to inflammatory stimuli was determined by real time PCR.
The NOD2 gene spans 39 KB and contains 12 coding exons on chromosome 8. Expression of mouse NOD2 into HEK cells resulted in NF-kappaB activation. NOD2 was found to be expressed in all mouse tissues analyzed except skin, with highest levels in lung, thymus and spleen. NOD2 mRNA levels increased greater than two-fold in a monocyte cell line in response to lipopolysaccharide, lipoteichoic acid, interferon-g and tumor necrosis factor-alpha.
Common structural and functional features between human and mouse NOD2 were identified. This should allow for development of relevant animal models to evaluate the role of NOD2 in chronic inflammatory disorders.
Inflammation Research 07/2003; 52(6):272-6. · 2.11 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Liver tissue from hepatitis C virus (HCV)-related end-stage disease contains T-cell infiltrates. The goal of this study is to determine whether CD4 T-cell clones established in vitro using an antigen-independent technique from explanted liver tissue (n = 3) are representative of dominant clones present in vivo. T-cell receptor (TCR) use by intrahepatic CD4 T cells was assessed by spectratype analysis. Clones were established from single CD4 T cells by culturing in vitro with anti-CD3 and interleukin-2 (n > 25 per patient). TCR genes expressed by each clone were identified by sequencing. When identical clones were isolated, the original spectratype was analyzed further to determine whether the clone was a dominant T-cell expansion in vivo. Evidence for clonal expansions was found in all patients by spectratyping. T cells expressing the same TCRBV genes used for spectratyping were cloned in vitro. Duplicate clones expressing the same TCR genes were observed in 2 patients. Confirmation that clones established in vitro matched those present in vivo was obtained for 2 clones. Many dominant clones identified by spectratyping did not proliferate in vitro. Although spectratyping suggested the widespread accumulation of clonal expansions in HCV-related end-stage liver disease, clones established in vitro using anti-CD3 were poorly representative of dominant clones present in vivo. Although cloning with anti-CD3 has the advantage of generating T-cell clones not biased a priori toward a specific antigen, modified cloning strategies will need to be developed to expand those clones that appear dominant in end-stage organs.
Liver Transplantation 08/2001; 7(8):716-23. · 3.39 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Autoimmune diseases result from the activation of self-reactive T cells recognizing autoantigens or foreign antigens cross-reactive with an autoantigen. T cells are thought to play a major role in autoimmune diseases in different organs, including the eye. This review focuses on the role of T cells in autoimmune uveitis in humans and in animal models of experimental autoimmune uveitis. Since rheumatoid arthritis is an autoimmune disease that has been studied far more extensively than uveitis, we have also included a review of clinical and experimental observations relevant to that disease.
Ocular Immunology and Inflammation 07/2000; 8(2):93-100. · 1.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To review the role of gene products from the human leukocyte antigen (HLA) complex in the normal functioning of the immune system, ocular inflammation, and models of autoimmunity.
A review of recently published reports.
Many chronic ocular inflammatory diseases are associated with specific alleles of the HLA complex. Understanding how HLA gene products function normally provides clues to the mechanism of disease associations. In the thymus, these molecules control the shape of the developing T-cell repertoire, leading to self-tolerance. In the periphery, HLA molecules bind and present peptide fragments to T cells, leading to a variety of effector functions. Although effector functions are for the most part beneficial, models are reviewed in which peptide-HLA interactions lead to T-cell responses with pathologic consequences. Herpes stromal keratitis is an informative animal model highlighting the role of self-tolerance, infection, and molecular mimicry in the development of autoimmunity.
Human leukocyte antigen gene products may be associated with chronic inflammatory disorders through the unique presentation of "disease-inducing" peptides or the development of a T-cell repertoire prone to autoreactivity and molecular mimicry.
American Journal of Ophthalmology 03/2000; 129(2):235-43. · 4.22 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Agents that increase intracellular cAMP have been shown to reduce joint inflammation in experimental arthritis, presumably by lowering the release of proinflammatory cytokines, such as TNF-alpha. Recent studies suggest that, in joints of patients with rheumatoid arthritis, TNF-alpha release from macrophages is triggered by their interaction with IL-15-stimulated T lymphocytes. In this report, we analyze the effect of rolipram, a cAMP-specific phosphodiesterase inhibitor, on TNF-alpha production in this experimental system. Cocultures of U937 cells with IL-15-stimulated T cells, but not control T cells, resulted in increased release of TNF-alpha. Pretreatment of T cells with rolipram or cAMP analogues inhibited the IL-15-stimulated increases in proliferation, expression of cell surface molecules CD69, ICAM-1, and LFA-1, and release of TNF-alpha from macrophages. Addition of PMA to T cells dramatically increased the expression of cell surface molecules, but had little or no effect on TNF-alpha release from either T cells or from cocultures, suggesting that other surface molecules must also be involved in T cell/macrophage contact-mediated production of TNF-alpha. Addition of PMA synergistically increased the proliferation of IL-15-stimulated T cells and the secretion of TNF-alpha from IL-15-stimulated T cell/macrophage cocultures. Rolipram and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) blocked these increases. Measurement of protein kinase A (PKA) activity and the use of inhibitory cAMP analogues (RpCPT-cAMP) confirmed that rolipram worked by stimulating PKA. These data suggest that PKA-activating agents, such as rolipram, can block secretion of TNF-alpha from macrophages by inhibiting T cell activation and expression of surface molecules.
The Journal of Immunology 10/1999; 163(5):2836-43. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The ability to induce anergy in antigen-specific T cells has potential therapeutic value for altering pathologic immune responses. This study was undertaken to further analyze changes in cytokine production and intracellular signaling during anergy induction using high concentrations of native peptide ligand of tetanus toxoid (TT)- and myelin basic protein (MBP)-specific human T cell lines. The TT-selected T cell line could be rendered unresponsive to its dominant epitope in a dose-dependent manner (IC50 = 0.03 microg/ml). The TT-selected line, as well as three T cell clones established from this line, continued to produce IFN-gamma and significantly increased IL-4 and IL-10 production when anergy was induced with high concentrations of the immunodominant epitope. JNK enzymatic activity was blocked in anergized T cells. The MBP-selected line could likewise be rendered unresponsive by incubation with supraoptimal concentrations of immunodominant peptide and anergy induction was accompanied by IL-10 release. Both T cell lines could be anergized by the autopresentation of native peptide since anergy was induced in cultures lacking fresh antigen-presenting cells. This study shows that the mitogen-activated protein kinase cascade is blocked when anergy is induced to high concentrations of soluble peptide.
Cellular Immunology 10/1998; 188(2):125-36. · 1.97 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The identification of clonal T cells at sites of inflammation is hampered by the large number of polyclonal T cells that nonspecifically accumulate. In this report, we combine the use of T cell sorting with spectratyping of the third complementarity determining region (CDR3) and direct sequence analysis to rapidly screen for and identify clonal expansions of T cells from synovial tissue specimens from patients with rheumatoid arthritis (RA). Initially, we used a polymerase chain reaction specific for the variable region gene of the T cell receptor beta chain (TCRBV) to compare the TCRBV repertoire expressed by CD4+ T cells from the peripheral blood and synovium of five patients with long-standing RA. Each patient had several TCRBV genes that were amplified to a greater degree from synovium. Extensive sequence analysis (n > 170) showed that each patient contained junctional sequences that occurred more than once, implying the presence of T cell clones within the starting CD4+ T cell population. To assess a more straightforward approach to identifying clones, six additional patients were recruited and CD4+, TCRBV2+ synovial T cells were positively selected and analyzed by CDR3 spectratyping. Bands deviating from a normal distribution were excised from the gel and sequenced directly. Clones were detected in half of the patients. These data are consistent with the possibility of an antigen-driven T cell response in RA that remains present in the setting of advanced disease.
Human Immunology 06/1997; 55(1):11-21. · 2.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cyclic AMP-dependent protein kinase (PKA) is anchored at specific subcellular sites through the interaction of the regulatory subunit (R) with protein kinase A-anchoring proteins (AKAPs) via an amphipathic helix binding motif. Synthetic peptides containing this amphipathic helix domain competitively disrupt PKA binding to AKAPs and cause a loss of PKA modulation of cellular responses. In this report we use S-Ht31, a cell-permeant anchoring inhibitor peptide, to study the role of PKA anchoring in sperm. Our analysis of three species of mammalian sperm detected three isoforms of PKA (RIIalpha, RIIbeta, and RIbeta) and one 110-kDa AKAP. The addition of S-Ht31 to bovine caudal epididymal sperm inhibits motility in a time- and concentration-dependent manner. A control peptide, S-Ht31-P, identical to S-Ht31 except for a proline for isoleucine substitution to prevent amphipathic helix formation, had no effect on motility. The inhibition of motility by S-Ht31 is reversible but only if calcium is present in the suspension buffer, suggesting a role for PKA anchoring in regulating cellular calcium homeostasis. Surprisingly, inhibition of PKA catalytic activity had little effect on basal motility or motility stimulated by agents previously thought to work via PKA activation. These data suggest that the interaction of the regulatory subunit of PKA with sperm AKAPs, independent of PKA catalytic activity, is a key regulator of sperm motility and that disruption of this interaction using cell-permeable anchoring inhibitor peptides may form the basis of a sperm-targeted contraceptive.
Journal of Biological Chemistry 03/1997; 272(8):4747-52. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Understanding the structural features of naturally processed peptides found within the major histocompatibility complex (MHC) class II peptide binding groove from disease-associated MHC molecules may provide insights into the nature of potential disease-related antigens. Class II MHC/peptide complexes were purified by immunoaffinity from transformed B cell lines homozygous for DRB1*0404 (an allele associated with rheumatoid arthritis) and *0402 (a closely related allele not associated with this disease). Peptides were eluted at acidic pH, fractionated by reversed phase HPLC, and analyzed by capillary electrophoresis. Those fractions containing a single dominant peptide were sequenced by automated Edman degradation and tandem mass spectrometry. The predominant peptide species identified came from non-polymorphic regions of the HLA class I molecules expressed by each cell line. Peptides from DRB1*0404 were found to be nested clusters derived from positions 26-43 of the HLA-B and -C alpha-chain. DRB1*0402 contained as the predominant peptide species a nested cluster from positions 129-145 of the HLA-B alpha-chain. The primary structure of the class I derived peptides was consistent with that seen by peptides exhibiting promiscuous DR binding behavior. Processing of MHC-derived peptides by MHC class II molecules is a common occurrence in the transformed B cell lines analyzed.
Journal of Neuroscience Research 10/1996; 45(6):795-802. · 2.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: CD3+ large granular lymphocyte (LGL) leukemia is a disease of unknown etiology characterized by clonal proliferation of T cells that usually express T-cell receptor (TCR) alpha beta heterodimers. The purpose of this study was to identify the variable (V), joining (J), and diversity (D) region TCR beta-chain genes expressed by CD3+ LGL leukemic cells in an attempt to gain insights into the etiology of this disorder. Twelve patients with LGL leukemia were studied, including seven with both LGL leukemia and rheumatoid arthritis (RA). RA is also a disease of unknown etiology that occurs frequently in patients with LGL leukemia. Clonally expanded T cells that express specific TCR V beta genes have been identified in fluid and tissue specimens from the joints of patients with RA. In this study, V beta expression was determined by PCR using a panel of 22 unique V beta primers to amplify cDNA prepared from peripheral blood mononuclear cells (PBMC). A dominant V beta gene product was readily apparent in all patients. To confirm that the dominant V beta gene originated from a clonal expansion, DNA fragments corresponding to the dominant V beta genes were subcloned into plasmids and independently isolated recombinants were sequenced. V-D-J region sequences that occurred repeatedly indicated clonality. The V beta and J beta genes expressed by the leukemic cells showed a pattern of distribution that followed the frequency with which these genes are represented in the peripheral blood. The residues corresponding to the third complementarity-determining region of the TCR beta chain were different in all cases. A specific pattern of VDJ usage was not identified for those patients with both LGL leukemia and RA; however, utilization of V beta-6 by LGL clones (N = 3) was observed only in the setting of RA. These data suggest that leukemic CD3+ LGL cells have been clonally transformed in a random fashion with respect to the TCR beta chain.
Blood 02/1995; 85(1):146-50. · 9.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The peripheral T cell repertoire is shaped by positive and negative selection. These intrathymic events are dependent on the direct interaction of MHC and TCR molecules. Inasmuch as one possible mechanism for HLA-linked disease involves the role that these molecules play in shaping the peripheral T cell repertoire, an understanding of how stable the repertoire remains is an important question that will influence future studies. The purpose of this study was to analyze the stability of the T cell repertoire in monozygotic twins. To investigate this question the percentage of CD4 and CD8 T cells expressing TCR V beta gene products was determined for seven sets of healthy monozygotic twins ages 2 through 44. V beta expression was determined by three-color flow cytometric analysis using antibodies to V beta-5.1, -5.2, -5.3, -6.7, -8, and -12. The percentage of CD4 cells expressing each V beta gene was highly concordant between twins. In contrast, differences were noted for V beta expression within the CD8 subset. This was especially marked when sets of twins were studied (n = 3) where one individual had an underlying disease. Although expression in the CD4 subset was again concordant, significant differences were noted within the CD8 subset compared to the healthy twin. These data indicate that in both health and disease, the CD4 T cell repertoire is tightly regulated although often sizable differences have developed in the CD8 compartment.
The Journal of Immunology 02/1994; 152(1):315-21. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Conflicting data have been reported regarding the presence or absence of a predominant variable (V) region T-cell receptor (TCR) gene in the peripheral blood or synovial fluid of patients with rheumatoid arthritis (RA). In this study we have used the polymerase chain reaction (PCR) to compare the level of TCR V beta gene expression by peripheral blood mononuclear cells (PBMC) and by synovial fluid cells obtained from HLA DRB1 *0401 and *0404 RA patients. PCR was performed using cDNA synthesized from freshly obtained cells (not stimulated in vitro). The pattern of expression observed for most of the V beta genes studied showed either preferential expression by PBMC or similar levels of expression between PBMC and synovial fluid T-cells. However, among individual patients (N = 5), several V beta genes were identified that were expressed to a significantly greater degree by synovial fluid cells. V beta 14 expression was detected in PBMC of all patients and the level of transcripts encoding V beta 14 increased following stimulation with immobilized anti-CD3 and IL-2. In vitro manipulation of populations of T-cells was found to alter the level of expressed V beta gene products. A V beta gene common to all patients that was consistently deleted from PBMC or expressed to a greater degree by resident, unsorted synovial fluid cells compared to PBMC was not identified.
Clinical Immunology and Immunopathology 08/1993; 68(1):79-87.
-
[show abstract]
[hide abstract]
ABSTRACT: Studies in transgenic and inbred strains of mice have shown that the critical molecular interactions controlling positive selection involve major histocompatibility complex (MHC), T-cell receptor (TCR), and CD4 or CD8 coreceptor molecules. Correlations have been established between MHC gene products and the percentage of CD4 or CD8 T cells that express specific variable (V) beta-gene products as part of the alpha beta heterodimer. These studies have important implications regarding potential mechanisms of HLA-linked autoimmune diseases in humans. If similar interactions are required for positive selection in humans, one would predict that the TCR repertoire expressed by mature, peripheral blood CD4 and CD8 T cells would vary. To test this hypothesis the expression of specific TCR V beta-region genes by CD4 and CD8 T cells from healthy individuals was compared using both triple-color flow cytometry and polymerase chain reaction based experimental approaches. The results show that the TCR repertoire does vary as a function of CD4 and CD8 T-cell subsets. Among unrelated individuals certain V beta genes were consistently overrepresented in the CD4 population (V beta-5.1, -6.7a, and -18); some were skewed to the CD8 population (V beta-14) while others showed variable patterns (V beta-12 and -17). Deletion of entire V beta gene families was not observed suggesting that this is a rare event in humans. Attempts to correlate the expressed TCR repertoire in humans with HLA alleles will require consideration of these differences in expression as a function of subset.
Human Immunology 12/1991; 32(3):194-202. · 2.84 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Background: Uveitis is often associated with a systemic inflammatory disease such as ankylosing spondylitis. Our understanding of the eye's susceptibility to immune-mediated uveitis as in the apparent absence of infection has been limited by a relative lack of experimental models. Here we sought to assess whether ocular inflammation occurs in a previously described murine model of proteoglycan-induced spondylitis, wherein mice develop progressive spondylitis, sacroiliitis and peripheral arthritis - features common to the clinical presentations of ankylosing spondylitis. Methods: Using intravital microscopy we examined the ocular inflammatory response after the onset of arthritis in mice that overexpressed the T cell receptor (TCR) specific for a dominant arthritogenic epitope of cartilage proteoglycan [TCR-Tg (transgenic) mice] or BALB/c controls. Results: Immunized TCR-Tg mice showed a significant increase in the number of rolling and adhering cells within the iris vasculature compared to adjuvant control mice. Cellular infiltration within the iris tissue, as assessed by intravital microscopy and histology, was also increased. Our initial temporal analysis has revealed that immunized TCR-Tg mice show a significant increase in intravascular inflammation by 2 weeks after immunization, but it diminishes at 4 weeks after immunization. Conclusions: Although these data are preliminary, this model has the potential to clarify the mechanisms accounting for the coexistence of eye and sacroiliac inflammation as occurs in patients with ankylosing spondylitis.
