Satwant K. Narula

Type 1 T regulatory cells

Summary: Suppression by T regulatory (Tr) cells is essential for induction of tolerance. Many types of Tr cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which these cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell‐to‐cell interactions and/or the production of interleukin (IL)‐10 and transforming growth factor (TGF)‐β. Type‐1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL‐10 and TGF‐β. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL‐10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL‐15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL‐10 and TGF‐β. Further characterisation of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen.

CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and immune responses in mucosal tissue.

Chemokine-directed migration of leukocyte subsets may contribute to the qualitative differences between systemic and mucosal immunity. Here, we demonstrate that in mice lacking the chemokine receptor CCR6, dendritic cells expressing CD11c and CD11b are absent from the subepithelial dome of Peyers patches. These mice also have an impaired humoral immune response to orally administered antigen and to the enteropathic virus rotavirus. In addition, CCR6(-/-) mice have a 2-fold to 15-fold increase in cells of select T lymphocyte populations within the mucosa, including CD4+ and CD8+ alphabeta-TCR T cells. By contrast, systemic immune responses to subcutaneous antigens in CCR6(-/-) mice are normal. These findings demonstrate that CCR6 is a mucosa-specific regulator of humoral immunity and lymphocyte homeostasis in the intestinal mucosa.

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation.

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 Å resolution) and iNOS (2.25 Å resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.

Ubiquitous Transgenic Expression of the IL-23 Subunit p19 Induces Multiorgan Inflammation, Runting, Infertility, and Premature Death

p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-α and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.

Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines

Cloned T regulatory type 1 (Tr1) cells produce IL‐10, TGF‐β, IFN‐γ, and very low or non‐detectable levels of IL‐2 and IL‐4, following TCR‐mediated activation. In addition, upon TCR stimulation, Tr1 cell clones up‐regulate activation markers but show low proliferative responses, partially due to the suppressive effect of autocrine IL‐10 and TGF‐β. Here we show that Tr1 cells have growth requirements different from those of Th1 and Th2 cells. Exogenous IL‐15, and to a lesser extent IL‐2, induce and support the proliferation of Tr1 cells in the absence of TCR activation. This strong cytokine response correlates with high constitutive levels of the IL‐2/15Rβ and common γ chains expressed by Tr1 cell clones. Furthermore, suboptimal doses of IL‐15, in combination with IL‐2, induce a significant growth (median value: 25‐fold increase in cell number) of Tr1 cell clones during a culture period of 11 days, which leads to an in vitro expansion of Tr1 cell clones comparable to that of Th1 and Th2 cell clones. Tr1 cell clones cultured in IL‐15 continue to secrete immunosuppressive cytokines and to proliferate poorly upon reactivation via TCR. These findings indicate that Tr1 cells are constitutively capable of responding to cytokines and mainly to IL‐15. This growth factor enables a significant in vitro expansion of Tr1 cells facilitating further biological and biochemical characterization of this unique T cell subset.

Purification of ADAM 10 from bovine spleen as a TNFα convertase

We have purified a protease with characteristics of TNFα convertase from bovine spleen membranes. Peptide sequencing of the purified protein identified it as ADAM 10 (Genbank accession no. Z21961). This metalloprotease cleaves a recombinant proTNFα substrate to mature TNFα, and can cleave a synthetic peptide substrate to yield the mature TNFα amino terminus in vitro. The enzyme is sensitive to a hydroxamate inhibitor of MMPs, but insensitive to phosphoramidon. In addition, cloned ADAM 10 mediates proTNFα processing in a processing‐incompetent cell line.

Regulation of granulocyte colony-stimulating factor gene expression by interleukin-17.

Interleukin-17 (IL-17) has been previously reported to induce stromal cells to produce a number of hematopoietic and proinflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF). Here, we have evaluated the mechanisms responsible for the augmentation of G-CSF gene expression by IL-17, using the murine 3T3 fibroblast cell line. Treatment of 3T3 cells, but not primary bone marrow-derived macrophages or murine monocyte/macrophage cell lines, resulted in increased steady-state G-CSF mRNA levels within 2-4 h and augmented G-CSF protein production. The combination of IL-17 and LPS enhanced G-CSF expression in an additive fashion. Stability studies revealed that IL-17 stabilized G-CSF mRNA levels, with a t1/2 of 4 h, compared to a t1/2 of less than 2 h in medium or LPS-treated cells. Induction of G-CSF expression in 3T3 cells by IL-17 did not appear to require tyrosine kinase activation or de novo protein synthesis. These studies indicate that post-transcriptional mechanisms play an important role in IL-17-induced G-CSF expression in fibroblasts and suggest that IL-17 may be useful for further delineating mechanisms of G-CSF gene regulation.

Interleukin-10 Suppresses the Development of Collagen Type II-Induced Arthritis and Ameliorates Sustained Arthritis in Rats

The collagen‐induced arthritis model in DA rats induced with homologous rat type II collagen was chosen to determine the therapeutic capacity and effects on autoimmunity by IL‐10. Systemic IL‐10 treatment (100 or 10 μg/day) with mini‐osmotic pumps during the periods of arthritis onset (days 12–20 after immunization) decreased the frequency of arthritis and delayed the onset and reduced the severity of arthritis in the few rats that eventually developed arthritis. Concomitantly, levels of autoantibodies to CII were reduced. To test the activity on established arthritis, IL‐10 was administered subcutaneously in the paws. This treatment reduced the swelling but did not block the arthritis process. The effective treatment required 100 μg of IL‐10 every 12th hour while 50 μg of IL‐10 had little effect, although a tendency of reduced paw swelling was observed. Surprisingly, therapeutic IL‐10 treatment led to higher serum levels of autoantibodies to CII. The highest doses of IL‐10 (100 μg) did not show any apparent toxic effects when given locally or systematically. Taken together, this study suggests that IL‐10 is a candidate for treatment of rheumatoid arthritis.

RN486, a Selective Bruton's Tyrosine Kinase Inhibitor, Abrogates Immune Hypersensitivity Responses and Arthritis in Rodents

Genetic mutation and pharmacological inhibition of Brutons tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC50 = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC50 = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC50 = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.

Blocking ion channel KCNN4 alleviates the symptoms of experimental autoimmune encephalomyelitis in mice

The KCNN4 potassium‐ion channel has been reported to play an important role in regulating antigen‐induced T cell effector functions in vitro. This study presents the first evidence that a selective KCNN4 blocker, TRAM‐34, confers protection against experimental autoimmune encephalomyelitis (EAE) in the mouse model. Treatment with the KCNN4 blocker did not prevent infiltration of T cells in the spinal cord, but resulted in the reduction of both the protein and the message levels of TNF‐α and IFN‐γ as well as the message levels of several other pro‐inflammatory molecules in the spinal cord. Plasma concentrations of TRAM‐34 within a 24‐h period were between the in vitro IC50 and IC90 values for the KCNN4 channel. The effect of TRAM‐34 was reversible, as indicated by the development of clinical EAE symptoms within 48 h after withdrawal of treatment. In summary, our data support the idea that KCNN4 channels play a critical role in the immune response during the development of MOG‐induced EAE in C57BL/6 mice.