NOD2 downregulates colonic inflammation by IRF4-mediated inhibition of K63-linked polyubiquitination of RICK and TRAF6.

It is well established that polymorphisms of the caspase activation and recruitment domain 15 (CARD15) gene, a major risk factor in Crohn's disease (CD), lead to loss of nucleotide-binding oligomerization domain 2 (NOD2) function. However, a molecular explanation of how such loss of function leads to increased susceptibility to CD has remained unclear. In a previous study exploring this question, we reported that activation of NOD2 in human dendritic cells by its ligand, muramyl dipeptide (MDP), negatively regulates Toll-like receptor (TLR)-mediated inflammatory responses. Here we show that NOD2 activation results in increased interferon regulatory factor 4 (IRF4) expression and binding to tumor necrosis factor receptor associated factor 6 (TRAF6) and RICK (receptor interacting serine-threonine kinase). We then show that such binding leads to IRF4-mediated inhibition of Lys63-linked polyubiquitination of TRAF6 and RICK and thus to downregulation of nuclear factor (NF)-κB activation. Finally, we demonstrate that protection of mice from the development of experimental colitis by MDP or IRF4 administration is accompanied by similar IRF4-mediated effects on polyubiquitination of TRAF6 and RICK in colonic lamina propria mononuclear cells. These findings thus define a mechanism of NOD2-mediated regulation of innate immune responses to intestinal microflora that could explain the relation of CARD15 polymorphisms and resultant NOD2 dysfunction to CD.

Molecular mechanisms regulating TGF-beta-induced Foxp3 expression.

Molecular mechanisms regulating transforming growth factor-beta (TGF-beta) induction of Foxp3 (forkhead box P3) expression and thus induction of induced regulatory T cells (Tregs) have been the focus of a great deal of study in recent years. It has become clear that this process is influenced by a number of factors as perhaps might be predicted by the fact that there is an overarching need of the immune system to finetune response to environmental antigens. In this review we discuss these mechanisms, with the aim of presenting a broad picture of how the various observations fit together to form an integrated regulatory regime.

Regulatory T cells and the induction of IL-17.

T helper (Th)17 cells have been shown to play a role in the pathogenesis of inflammatory and autoimmune diseases including inflammatory bowel diseases (IBD). It is now well established that although transforming growth factor (TGF)-beta alone induces FoxP3(+) regulatory T (Treg) cells, TGF-beta and interleukin (IL)-6, acting in concert, induce differentiation of mouse naive T cells into Th17. As we previously showed that CD4(+)CD25(+)Foxp3(+) "natural" Treg cells express cell surface or secrete TGF-beta, we examined whether Treg cells serve to induce Th17 differentiation. We found that upon activation, Treg cells induce CD4(+)CD25(-) naive T cells or Treg cells themselves to differentiate into Th17 in the presence of IL-6 alone without exogenous addition of TGF-beta. We also found that TGF-â is also produced by dendritic cells that are in contact with Treg cells. Although Treg cells are effectively recruited at inflamed mucosa in patients with IBD, it is possible that Treg cells may have undesirable effects through their ability to differentiate into pathogenic Th17 in the presence of IL-6 and/or IL-23 at sites of inflammation. Further study of the relationship between Treg cells and Th17 cells in the inflamed tissue in IBD is important for possible Treg cell-mediated therapeutic applications.

The molecular basis of NOD2 susceptibility mutations in Crohn's disease.

Nucleotide oligomerization domain (NOD)2 is a member of the NOD-like receptor family of proteins that initiate inflammatory responses when exposed to ligands derived from bacterial components that gain access to the intracellular milieu. It is thus somewhat paradoxical that polymorphisms in the gene that encode NOD2 (CARD15) that lead to impaired NOD2 function, are susceptibility factors in Crohn's disease, a condition marked by excessive inflammatory responses to normal bacterial flora. In an initial series of studies conducted in our laboratory to better define NOD2 function and to resolve this paradox we showed that NOD2 activation by its ligand, muramyl dipeptide (MDP) ordinarily downregulates responses to Toll-like receptor (TLR) stimulation, and thus cells lacking NOD2 mount increased responses to such stimulation. This fits with the fact that mice bearing an NOD2 transgene, and thus having cells with increased NOD2 function display decreased responses to TLR stimulation and are resistant to experimental colitis induction. In further studies, we showed that prestimulation of cells with NOD2 ligand renders them unresponsive to TLR stimulation, because such prestimulation results in the elaboration of inhibitory factor (IRF4), an inhibitor of TLR-induced inflammatory pathways. Furthermore, administration of MDP to normal mice induces IRF4 and prevents experimental colitis. These studies strongly suggest that NOD2 polymorphisms are associated with Crohn's disease because they lead to a decrease in the negative regulation of TLR responses occurring in the normal gut, and thus a pathologic increase in responses to the normal flora. The finding that MDP administration prevents experimental colitis opens the door to the possibility that such treatment might quell Crohn's disease relapses in patients without NOD2 abnormalities.

Cell contact-dependent immunosuppression by CD4(+)CD25(+) regulatory T cells is mediated by cell surface-bound transforming growth factor beta.

CD4(+)CD25(+) T cells have been identified as a population of immunoregulatory T cells, which mediate suppression of CD4(+)CD25(-) T cells by cell-cell contact and not secretion of suppressor cytokines. In this study, we demonstrated that CD4(+)CD25(+) T cells do produce high levels of transforming growth factor (TGF)-beta1 and interleukin (IL)-10 compared with CD4(+)CD25(-) T cells when stimulated by plate-bound anti-CD3 and soluble anti-CD28 and/or IL-2, and secretion of TGF-beta1 (but not other cytokines), is further enhanced by costimulation via cytotoxic T lymphocyte-associated antigen (CTLA)-4. As in prior studies, we found that CD4(+)CD25(+) T cells suppress proliferation of CD4(+)CD25(-) T cells; however, we observed here that such suppression is abolished by the presence of anti-TGF-beta. In addition, we found that CD4(+)CD25(+) T cells suppress B cell immunoglobulin production and that anti-TGF-beta again abolishes such suppression. Finally, we found that stimulated CD4(+)CD25(+) T cells but not CD4(+)CD25(-) T cells express high and persistent levels of TGF-beta1 on the cell surface. This, plus the fact that we could find no evidence that a soluble factor mediates suppression, strongly suggests that CD4(+)CD25(+) T cells exert immunosuppression by a cell-cell interaction involving cell surface TGF-beta1.

Regulation of experimental mucosal inflammation.

Studies conducted over the past 10 years have provided ample evidence that many types of inflammations arising from basic abnormalities of immune regulation are ultimately 'funneled' through a Th1 or Th2 T cell-mediated immune reaction. Thus, by understanding these types of reactions and, in particular, by identifying their natural checkpoints, one can control the inflammation regardless of its more basic causes. A case in point is the inflammatory disease of the intestine known as Crohn disease, a disease now thought to be due to one or more abnormalities leading to an excessive immune response to elements of the bacterial microflora of the gut. Both in murine models and by study of Crohn disease itself, we have shown that Crohn inflammation is due to a Th1 T-cell abnormality involving overproduction of interleukin (IL)-12, interferon (IFN-gamma, and tumor necrosis factor (TNF)-alpha. In addition, we and others have shown that treatment of mice with anti-IL-12 or other agents that downregulate the level of IL- 12 secretion can have a dramatic effect on the inflammation. This is because anti-IL-12 administration leads to apoptosis of activated Th1 T cells. A second checkpoint of Th1 T-cell-mediated inflammation involves its downregulation by the suppressor cytokine, transforming growth factor (TGF)-beta. We have been delivering TGF-beta to mice with experimental intestinal inflammation, using several novel approaches. In particular, we have successfully treated such mice with intranasally administered DNA encoding active TGF-beta. Another approach currently under investigation is delivery of TGF-beta by gene therapy. These and other developments in the understanding of inflammation paint a bright future for cytokine-based therapeutic agents. It is now apparent that these therapies are not only effective and safe but also potentially long-lasting.

Treatment of experimental (Trinitrobenzene sulfonic acid) colitis by intranasal administration of transforming growth factor (TGF)-beta1 plasmid: TGF-beta1-mediated suppression of T helper cell type 1 response occurs by interleukin (IL)-10 induction and IL-12 receptor beta2 chain downregulation.

In this study, we show that a single intranasal dose of a plasmid encoding active transforming growth factor beta1 (pCMV-TGF-beta1) prevents the development of T helper cell type 1 (Th1)-mediated experimental colitis induced by the haptenating reagent, 2,4, 6-trinitrobenzene sulfonic acid (TNBS). In addition, such plasmid administration abrogates TNBS colitis after it has been established, whereas, in contrast, intraperitoneal administration of rTGF-beta1 protein does not have this effect. Intranasal pCMV-TGF-beta1 administration leads to the expression of TGF-beta1 mRNA in the intestinal lamina propria and spleen for 2 wk, as well as the appearance of TGF-beta1-producing T cells and macrophages in these tissues, and is not associated with the appearances of fibrosis. These cells cause marked suppression of interleukin (IL)-12 and interferon (IFN)-gamma production and enhancement of IL-10 production; in addition, they inhibit IL-12 receptor beta2 (IL-12Rbeta2) chain expression. Coadministration of anti-IL-10 at the time of pCMV-TGF-beta1 administration prevents the enhancement of IL-10 production and reverses the suppression of IL-12 but not IFN-gamma secretion. However, anti-IL-10 leads to increased tumor necrosis factor alpha production, especially in established colitis. Taken together, these studies show that TGF-beta1 inhibition of a Th1-mediated colitis is due to: (a) suppression of IL-12 secretion by IL-10 induction and (b) inhibition of IL-12 signaling via downregulation of IL-12Rbeta2 chain expression. In addition, TGF-beta1 may also have an inhibitory effect on IFN-gamma transcription.

Activated self-MHC-reactive T cells have the cytokine phenotype of Th3/T regulatory cell 1 T cells.

In the present study, we show that human self-MHC-reactive (autoreactive) T cell clones are functionally distinct from Ag-specific T cell clones. Self-MHC-reactive T cells exhibited helper function for B cell Ig production when cultured with non-T cells alone, and they exhibit suppressor function when cultured with PWM- or rCD40 ligand (rCD40L)-activated non-T cells, whereas tetanus toxoid (TT)-specific clones exhibited only helper function in the presence of TT with or without PWM or rCD40L. Addition of neutralizing Abs to the cultures showed that the suppression was mediated by TGF-beta but not by IL-10 or IFN-gamma. The self-MHC-reactive clones also inhibited proliferation of primary CD4+ T cells and TT-specific T cell clones, but in this case the inhibition was mediated by both IL-10 and TGF-beta. In further studies, the interactions between self-MHC-reactive T cell clones and non-T cells that led to suppressor cytokine production have been explored. We found that prestimulation of non-T cells for 8 h with PWM or for 48 h for rCD40L results in non-T cells capable of inducing self-MHC-reactive T cell to produce high levels of TGF-beta and IL-10. In addition, these prestimulation times coincided with peak induction of HLA-DR and costimulatory B7 molecule (especially CD86) expression on B cells. Finally, addition of CTLA-4/Fc or blocking F(ab')2 anti-CTLA-4 mAb, plus optimally stimulated non-T cells, to cultures of self-MHC-reactive clones inhibited the induction of TGF-beta but not IL-10 or IFN-gamma production. In summary, these studies show that activated self-MHC-reactive T cells have the cytokine phenotype of Th3 or T regulatory cell 1 and thus may be important regulatory cells that mediate oral and peripheral tolerance and prevent the development of autoimmunity.

Soluble VCAM-1 induces chemotaxis of Jurkat and synovial fluid T cells bearing high affinity very late antigen-4.

It has been shown that cells with high affinity very late Ag (VLA)-integrins have up-regulated expression of a beta1-subunit epitope, which is detected by 15/7 mAb. In this study, we demonstrate that soluble VCAM-1 (sVCAM-1) exhibits chemotactic activity of T cells with high affinity VLA-4 against VCAM-1, such as Jurkat T cells and IL-2-dependent T cells. Moreover, we found that T cells in the synovial fluid show high basal migration in the absence of sVCAM-1, compared with peripheral blood T cells in patients with rheumatoid arthritis. Among T cells in the synovial fluid, CD45RO+ memory T cells, in response to sVCAM-1, showed a much higher than basal migratory response when compared with CD45RA+ naive cells, while no significant difference was observed between CD4+ and CD8+ T cells. The chemotactic activity of sVCAM-1 is inhibited in the presence of anti-VCAM-1 and anti-VLA-4, which interfered with the binding between VCAM-1 and VLA-4. Inhibition studies using various kinase inhibitors (C3 exoenzyme, KN62, and H7) show that Rho, Ca2+/calmodulin-dependent kinase II, and protein kinase C are involved in signal transduction in sVCAM-1-induced chemotaxis, respectively, whereas tyrosine kinase seems to play a lesser role, since genistein showed only partial inhibition of T cell chemotaxis. Western blot analysis using an anti-phospho-serine mAb (MO82) reveals that Ser82 in the vimentin is phosphorylated specifically by Ca2+/calmodulin-dependent kinase II through sVCAM-1 activation in the IL-2 dependent T cells. Collectively, by inducing migration and recruitment of T cells through several kinase activations, sVCAM-1 contributes to the development of the inflammation of synovial lesion.

Expression of interleukin-18 in murine contact hypersensitivity.

In this study, we made a mouse model for contact hypersensitivity (CH) using oxazolone as a contact allergen and examined the expression of interleukin-18 (IL-18) in the diseased skin sites at both mRNA and protein levels. In the kinetic study by semiquantitative RT-PCR, IL-18 mRNA was constitutively produced in normal murine skin but increased significantly at 12 h and peaked at 24 h in the ear skin of CH mice. A positive correlation was confirmed between the IL-18 mRNA signal and CH, as measured by mouse ear swelling response. Histologically, in situ hybridization showed that the IL-18 mRNA signal was weakly observed in the dermis but not the epidermis of normal skin, whereas the IL-18 mRNA signal was found intensively in the dermis, particularly in inflammatory cell areas. Using IL-18-specific antibody immunostaining, it was further found that IL-18 protein production had a histologic location similar to that of IL-18 mRNA in both normal and CH mice. The present study suggests that IL-18 may be implicated in the pathogenesis of murine CH.

Interleukin-12 enhances contact hypersensitivity by modulating the in vivo cytokine pattern in mice.

It has been proven that interleukin-12 (IL-12) can modify Th1 and Th2 cell-mediated immune diseases by altering the development and cytokine production of the cells. In this study, we investigated the in vivo immunomodulatory effect of recombinant murine IL-12 on contact hypersensitivity, a Th1 cell-mediated disease. For this purpose, Balb/C mice were sensitized with 3% 4-ethyoxymethylene-2-phenyl-oxazol-5-one (OXAZ), and recombinant mouse IL-12 was given simultaneously during the induction phase. Contact allergy was then elicited by ear challenge with 1% OXAZ. We examined the mouse ear swelling response, in vivo cytokine gene expression in the skin and local lymph nodes, and in vitro cytokine production by the spleen lymphocytes. It was found that in vivo IL-12 treatment during the induction phase significantly enhanced the ear swelling response to OXAZ in sensitized mice. Moreover, remarkable mononuclear cell infiltration and edema and higher expression of Th1 cytokine mRNAs (IL-2 and interferon-gamma) in the skin lesion and local lymph nodes were observed in contact allergic mice with IL-12 treatment compared with contact allergic mice without IL-12 treatment. The expression of Th2 cytokine mRNA (IL-4) in the skin lesion and local lymph nodes, however, was largely downregulated, with no change in IL-5 mRNA in IL-12-treated contact allergic mice. We found, unexpectedly, that, similar to the effects on phytohemagglutinin (PHA) stimulated in vitro IL-2 and IFN-gamma production, PHA-induced in vitro IL-4 production was enhanced in the spleen lymphocytes from IL-12-treated contact allergic mice. Our results indicate that exogenous IL-12 enhanced contact hypersensitivity probably because of the in vivo promoting and suppressing effects of IL-12 on Th1 and Th2 gene expression, respectively.

The role of VCAM-1 molecule in the pathogenesis of rheumatoid synovitis.

The present review focuses on the possible role of VCAM-1 expression on synovial fibroblast-like cells in the synovial lesion of rheumatoid arthritis (RA). The VCAM-1 expressing cells were mainly present in the synovial lining layer. The VCAM-1 expressing fibroblast-like cells also showed activity of uridine diphosphoglucose dehydrogenase, indicating that they are activated fibroblasts. VCAM-1 expressing T cells were also found in RA synovial fluids, where T lymphocytes show upregulation of alpha 4 beta 1 expression, and these T lymphocytes are able to bind to VCAM-1 in solid phase. Further experiments excluded the production of VCAM-1 protein in synovial fluid T lymphocytes and supported the idea that the soluble VCAM-1 was bound to the surface of synovial fluid T lymphocytes. We next planned to examine the effect of soluble VCAM-1 on T cell functions, by using recombinant soluble VCAM-1. The recombinant soluble VCAM-1 rendered synovial or peripheral T cells anergic to various stimuli. These findings imply that recombinant soluble VCAM-1 might be useful as a therapeutic tool to prevent abnormal immune response, since it binds to activated T lymphocytes with upregulation of alpha 4 beta 1, but not to resting T lymphocytes, and soluble VCAM-1 bound T lymphocytes become anergic.

T cells bound by vascular cell adhesion molecule-1/CD106 in synovial fluid in rheumatoid arthritis: inhibitory role of soluble vascular cell adhesion molecule-1 in T cell activation.

Elevated levels of soluble vascular cell adhesion molecule-1 (sVCAM-1)/CD106 have been reported in synovial fluid (SF) from patients with rheumatoid arthritis (RA). In the present study, VCAM-1-positive lymphocytes were found in SF from RA patients. The data strongly suggest that sVCAM-1 might be bound to lymphocytes in SF. rsVCAM-1 in the fluid phase can bind to both SF lymphocytes and IL-2-dependent T cell lines with up-regulated expression and binding activity of VLA-4. Furthermore, proliferative responses of SF mononuclear cells (SFMC) with PHA, immobilized anti-CD3, or anti-CD2 and PMA were inhibited to various extents in the presence of rsVCAM-1, but only PMA-induced proliferative response of PBMC from normal individuals was inhibited notably in the presence of rsVCAM-1. rsVCAM-1 also drastically reduced IL-2 production of Jurkat leukemic T cells possessing high affinity VLA-4 with the stimulation of anti-CD3 and PMA, suggesting that the T cell hyporesponsiveness induced by rsVCAM-1 might stem from impairment of IL-2 production. These results indicate that sVCAM-1 provides a negative signal to T cell activation, probably by affecting the pathway of protein kinase C activation. Thus, binding of sVCAM-1 to SF lymphocytes might partly explain the anergic state of these lymphocytes.

Differential regulation of C alpha 1 and C alpha 2 germ-line and mature mRNA transcripts in human peripheral blood B cells.

In this study, we investigated the regulation of germ-line and mature C alpha mRNA transcript expression in human peripheral blood B cells. In initial studies, we found that C alpha germ-line and mature transcripts were constitutively expressed in total peripheral blood B cells but not in high density surface IgM+ (surface IgA-) B cells; the latter cells were therefore used in subsequent studies. Focusing first on regulation of germ-line (I alpha-C alpha) transcripts, we showed that C alpha 1 germ-line transcripts are induced by TGF-beta 1 alone but such induction is enhanced by Staphylococcus aureus, Cowan I (SAC). In contrast, C alpha 2 germ-line transcripts are optimally induced by TGF-beta 1 in the absence of a B cell stimulus. In addition, although SAC alone induced both C alpha 1 and C alpha 2 germ-line transcripts, such induction is dependent on endogenous (B cell-derived) TGF-beta 1 production, because no induction is detected in cells cultured in the presence of neutralizing anti-TGF-beta 1. Turning next to mature (VDJ-C alpha) transcripts we showed that SAC plus TGF-beta 1 induces mature C alpha 1 transcripts, and such induction is IL-10 dependent because it is enhanced by exogenous IL-10 and abrogated by the presence of anti-IL-10. In contrast, SAC plus TGF-beta 1 does not induce C alpha 2 mature transcripts, even in the presence of exogenous IL-10; such transcripts are induced, however, by T cell stimuli such as anti-CD40 (presented by CDw32-transfected L cells) in the presence of TGF-beta 1/IL-10 or by PWM-activated T cells. In summary, these studies show that C alpha 1 and C alpha 2 germ-line transcript induction is differentially regulated, in keeping with previous studies of differences in germ-line CH transcript induction in the first and second IgH duplication units. Furthermore, C alpha 1 and C alpha 2 mature transcript induction is also differentially regulated, with C alpha 2 requiring a T cell signal such as that delivered by the CD40 ligand. Finally, IL-10 appears to be uniquely supportive of the induction of both C alpha 1 and C alpha 2 mature transcripts.

Regulation of C gamma subclass germ-line transcripts in human peripheral blood B cells.

In the present study we investigated the early steps of human IgG subclass differentiation by defining the conditions necessary for IgG subclass-specific production of germ-line transcripts in human peripheral blood B cells. Constant region of gamma-globulin (C gamma) subclass germ-line transcripts were measured using newly developed reverse transcription and polymerase chain reaction (RT-PCR) assays that were shown to be C gamma subclass-specific germ-line transcripts by size of the amplification products obtained before and after digestion with appropriate endonucleases (NarI and NcoI). In initial studies we found that C gamma 1 and C gamma 2 germ-line transcripts were constitutively expressed in total peripheral blood B cells, but not in high density sIgM+(sIgG-) B cells prepared with Percoll density gradients and magnetic beads separation techniques; the latter cells were therefore used throughout this study. Induction of germ-line transcripts (germ-line C gamma 1 transcript) was noted in stimulated B cells (SAC plus IL-2) at 6 h; thus, the appearance of germ-line transcripts could not be attributed to preferential growth of B cells expressing germ-line transcripts. In subsequent studies we found that induction of germ-line transcripts for the various IgG subclasses fell into two patterns. Induction of C gamma 3 and C gamma 1 germ-line transcripts, transcripts of genes in the first human duplication unit, generally required a proliferative stimulus (Staphylococcus aureus Cowan I, SAC) and was brought about by SAC plus IL-4 (C gamma 3 germ-line transcripts) and SAC alone or SAC plus IL-2 (C gamma 1 germ-line transcripts); in contrast, induction of C gamma 2 and C gamma 4 germ-line transcripts, transcripts of genes in the second duplication unit, was accomplished with cytokines alone: IFN-gamma (C gamma 2 germ-line transcripts) and IL-4 (C gamma 4 germ-line transcripts), and was not augmented by addition of a proliferative stimulus. Finally, we found that IFN-gamma inhibited IL-4-induced C gamma 3 and C gamma 4 germ-line transcripts (with or without SAC). These findings establish that the various human IgG subclasses manifest distinct requirements for the regulation of early steps in isotype differentiation. In addition, they suggest that human C gamma genes exhibit patterns of regulation related to their respective duplication units.

Plasma thrombomodulin and alpha 2-plasmin inhibitor-plasmin complex are elevated in active systemic lupus erythematosus.

Plasma levels of thrombomodulin and alpha 2-plasmin inhibitor-plasmin complex were measured by ELISA in patients with rheumatic diseases. Thrombomodulin levels in patients with active systemic lupus erythematosus (SLE) were significantly higher than those in patients with inactive SLE or in healthy controls. This suggests that thrombomodulin, normally a component of vascular endothelial cell membrane, is easily released to plasma in patients with active SLE. High titers of the thrombomodulin level and the correlated alpha 2-plasmin inhibitor-plasmin complex elevations imply vascular injury, and consequently, excessive fibrinolytic processes in active SLE.

Effect of prolyl 4-hydroxylase inhibitor on fibroblast collagen production in vitro: an approach to the treatment of systemic sclerosis.

Prolyl 4-hydroxylase (PH), which catalyzes the conversion of prolyl residues to 4-hydroxyproline, plays a central role in the synthesis and secretion of collagen. We demonstrated that fibroblasts from patients with systemic sclerosis produced significantly more PH than those from healthy subjects. To regulate excessive collagen production by scleroderma fibroblasts, we examined the effect of a PH inhibitor on collagen production by scleroderma fibroblasts. Fibrostatin-C, a PH inhibitor, significantly decreased the amount of procollagen (type I) production by scleroderma fibroblasts (p < 0.01). We suggest that this PH inhibitor might be a valid antifibrotic agent useful for the treatment of patients with scleroderma.