bluuebunny/crossref_metadata_2025 huggingface dataset:
Class Ia MHC-Deficient BALB/c Mice Generate CD8+ T Cell-Mediated Protective Immunity Against Listeria monocytogenes Infection
Authors: D’Orazio Sarah E. F. , Halme Dina G. , Ploegh Hidde L. , Starnbach Michael N.
Published: April 2014

CD8+ T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8+ T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The Kb−/−Db−/− mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 Kb−/−Db−/− mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive Kb−/−Db−/− mice indicated that CD8+ T cells were the major component of this protective immune response. A CD8+ T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-γ secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.
NAD(P)H Oxidase 1, a Product of Differentiated Colon Epithelial Cells, Can Partially Replace Glycoprotein 91phox in the Regulated Production of Superoxide by Phagocytes
Authors: Geiszt Miklós , Lekstrom Kristen , Brenner Sebastian , Hewitt Stephen M. , Dana Raya , Malech Harry L. , Leto Thomas L.
Published: April 2014

Reactive oxygen species (ROS) serve several physiological functions; in some settings they act in host defense, while in others they function in cellular signaling or in biosynthetic reactions. We studied the expression and function of a recently described source of ROS, NAD(P)H oxidase 1 or Nox1, which has been associated with cell proliferation. In situ hybridization in mouse colon revealed high Nox1 expression within the lower two-thirds of colon crypts, where epithelial cells undergo proliferation and differentiation. Human multitumor tissue array analysis confirmed colon-specific Nox1 expression, predominantly in differentiated epithelial tumors. Differentiation of Caco2 and HT29 cells with 1α,25-dihydroxyvitamin D3 or IFN-γ enhances Nox1 expression and decreases cell proliferation, suggesting that Nox1 does not function as a mitogenic oxidase in colon epithelial cells. Transduction with retrovirus encoding Nox1 restored activation and differentiation-dependent superoxide production in gp91phox-deficient PLB-985 cells, indicating close functional similarities to the phagocyte oxidase (phox). Furthermore, coexpression of cytosolic components, p47phox and p67phox, augments Nox1 activity in reconstituted K562 cells. Finally, Nox1 partially restores superoxide production in neutrophils differentiating ex vivo from gp91phox-deficient CD34+ peripheral blood-derived stem cells derived from patients with X-linked chronic granulomatous disease. These studies demonstrate a significant functional homology (cofactor-dependent and activation-regulated superoxide production) between Nox1 and its closest homologue, gp91phox, suggesting that targeted up-regulation of Nox1 expression in phagocytic cells could provide a novel approach in the molecular treatment of chronic granulomatous disease.
Greater CD8+ TCR Heterogeneity and Functional Flexibility in HIV-2 Compared to HIV-1 Infection
Authors: Lopes A. Ross , Jaye Assan , Dorrell Lucy , Sabally Sehu , Alabi Abraham , Jones Nicola A. , Flower Darren R. , De Groot Anne , Newton Phillipa , Lascar R. Monica , Williams Ian , Whittle Hilton , Bertoletti Antonio , Borrow Persephone , Maini Mala K.
Published: April 2014

Virus-specific CD8+ T cells are known to play an important role in the control of HIV infection. In this study we investigated whether there may be qualitative differences in the CD8+ T cell response in HIV-1- and HIV-2-infected individuals that contribute to the relatively efficient control of the latter infection. A molecular comparison of global TCR heterogeneity showed a more oligoclonal pattern of CD8 cells in HIV-1- than HIV-2-infected patients. This was reflected in restricted and conserved TCR usage by CD8+ T cells recognizing individual HLA-A2- and HLA-B57-restricted viral epitopes in HIV-1, with limited plasticity in their response to amino acid substitutions within these epitopes. The more diverse TCR usage observed for HIV-2-specific CD8+ T cells was associated with an enhanced potential for CD8 expansion and IFN-γ production on cross-recognition of variant epitopes. Our data suggest a mechanism that could account for any possible cross-protection that may be mediated by HIV-2-specific CD8+ T cells against HIV-1 infection. Furthermore, they have implications for HIV vaccine development, demonstrating an association between a polyclonal, virus-specific CD8+ T cell response and an enhanced capacity to tolerate substitutions within T cell epitopes.
Plasmodium chabaudi chabaudi Infection in Mice Induces Strong B Cell Responses and Striking But Temporary Changes in Splenic Cell Distribution
Authors: Achtman Ariel H. , Khan Mahmood , MacLennan Ian C. M. , Langhorne Jean
Published: April 2014

B cells and Abs play a key role in controlling the erythrocytic stage of malaria. However, little is known about the way the humoral response develops during infection. We show that Plasmodium chabaudi chabaudi causes major, but temporary changes in the distribution of leukocytes in the spleen. Despite these changes, an ordered response to infection develops, which includes vigorous extrafollicular growth of plasmablasts and germinal center formation. Early in the response, the lymphocytes in the T zone and follicles become widely spaced, and the edges of these compartments blur. This effect is maximal around the peak of parasitemia. Germinal centers are apparent by day 8, peak at day 20, and persist through day 60. Extrafollicular foci of plasmablasts are visible from day 4 and initiate a very strong plasma cell response. Initially, the plasma cells have a conventional red pulp distribution, but by day 10 they are unconventionally sited in the periarteriolar region of the white pulp. In this region they form clusters occupying part of the area normally filled by T cells. B cells are absent from the marginal zone for at least 30 days after the peak of infection, although flow cytometry shows their continued presence in the spleen throughout infection. Relatively normal splenic architecture is regained by day 60 of infection. These results show that the changes in splenic cell distribution are linked to the presence of parasites and do not seem to interfere with the development of the humoral response.
Cutting Edge: Link Between Innate and Adaptive Immunity: Toll-Like Receptor 2 Internalizes Antigen for Presentation to CD4+ T Cells and Could Be an Efficient Vaccine Target
Authors: Schjetne Karoline W. , Thompson Keith M. , Nilsen Nadra , Flo Trude H. , Fleckenstein Burkhard , Iversen Jens-Gustav , Espevik Terje , Bogen Bjarne
Published: April 2014

An ideal vaccine for induction of CD4+ T cell responses should induce local inflammation, maturation of APC, and peptide loading of MHC class II molecules. Ligation of Toll-like receptor (TLR) 2 provides the first two of these three criteria. We have studied whether targeting of TLR2 results in loading of MHC class II molecules and enhancement of CD4+ T cell responses. To dissociate MHC class II presentation from APC maturation, we have used an antagonistic, mouse anti-human TLR2 mAb (TL2.1) as ligand and measured proliferation of a mouse Cκ-specific human CD4+ T cell clone. TL2.1 mAb was 100-1000 times more efficiently presented by APC compared with isotype-matched control mAb. Moreover, TL2.1 mAb was internalized into endosomes and processed by the conventional MHC class II pathway. This novel function of TLR2 represents a link between innate and adaptive immunity and indicates that TLR2 could be a promising target for vaccines.
A Single Intratracheal Dose of the Growth Factor Fms-Like Tyrosine Kinase Receptor-3 Ligand Induces a Rapid Differential Increase of Dendritic Cells and Lymphocyte Subsets in Lung Tissue and Bronchoalveolar Lavage, Resulting in an Increased Local Antibody Production
Authors: Pabst Reinhard , Lührmann Anke , Steinmetz Ivo , Tschernig Thomas
Published: April 2014

Repetitive doses of the growth factor Fms-like tyrosine kinase receptor-3 ligand (Flt3L) have resulted in increased numbers of dendritic cells (DC) in various organs, and the effect on protective or tolerogeneic responses in the gut wall has been documented in the literature. In this study, for the first time, Flt3L was locally applied in the trachea of rats using a single dose only. A dose-dependent increase not only of DC, but also of T lymphocytes (CD4+ and CD8+), was seen with a maximum on day 3. The effects on the cells in the lung interstitium and the bronchoalveolar space showed some differences. The use of tetanus toxoid as a model Ag applied intratracheally after the local Flt3L stimulation resulted in increased levels of specific IgA and IgG in the lung. Thus, this novel approach of locally stimulating APCs by topical application of a DC growth factor before applying the Ag offers a new vaccination strategy.
Identification of A3 Receptor- and Mast Cell-Dependent and -Independent Components of Adenosine-Mediated Airway Responsiveness in Mice
Authors: Tilley Stephen L. , Tsai Mindy , Williams Cara M. , Wang Z.-S. , Erikson Christopher J. , Galli Stephen J. , Koller Beverly H.
Published: April 2014

Adenosine-induced bronchoconstriction is a well-recognized feature of atopic asthma. Adenosine acts through four different G protein-coupled receptors to produce a myriad of physiological effects. To examine the contribution of the A3 adenosine receptor to adenosine-induced bronchoconstriction and to assess the contribution of mast cells to this process, we quantified airway responsiveness to aerosolized adenosine in wild-type, A3 receptor-deficient, and mast cell-deficient mice. Compared with the robust airway responses elicited by adenosine in wild-type mice, both A3-deficient and mast cell-deficient mice exhibited a significantly attenuated response compared with their respective wild-type controls. Histological examination of the airways 4 h after adenosine exposure revealed extensive degranulation of airway mast cells as well as infiltration of neutrophils in wild-type mice, whereas these findings were much diminished in A3-deficient mice and were not different from those in PBS-treated controls. These data indicate that the airway responses to aerosolized adenosine in mice occur largely through A3 receptor activation and that mast cells contribute significantly to these responses, but that activation of additional adenosine receptors on a cell type(s) other than mast cells also contributes to adenosine-induced airway responsiveness in mice. Finally, our findings indicate that adenosine exposure can result in A3-dependent airway inflammation, as reflected in neutrophil recruitment, as well as alterations in airway function.
Activation of Murine Lung Mast Cells by the Adenosine A3 Receptor
Authors: Zhong Hongyan , Shlykov Sergiy G. , Molina Jose G. , Sanborn Barbara M. , Jacobson Marlene A. , Tilley Stephen L. , Blackburn Michael R.
Published: April 2014

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A2A, A2B, and A3 adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A3 receptors could induce mast cell histamine release in association with increases in intracellular Ca2+ that were mediated through Gi and phosphoinositide 3-kinase signaling pathways. The function of A3 receptors in vivo was tested by exposing mice to the A3 receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice while having no effect in A3 receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A3 receptors. These results demonstrate that the A3 adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.
Captopril Prevents Experimental Autoimmune Myocarditis
Authors: Godsel Lisa M. , Leon Juan S. , Wang Kegiang , Fornek Jamie L. , Molteni Agostino , Engman David M.
Published: April 2014

Captopril, an angiotensin-converting enzyme inhibitor, is widely used in the treatment of a variety of cardiomyopathies, but its effect on autoimmune myocarditis has not been addressed experimentally. We investigated the effect of captopril on myosin-induced experimental autoimmune myocarditis. A/J mice, immunized with syngeneic cardiac myosin, were given 75 mg/L of captopril in their drinking water. Captopril dramatically reduced the incidence and severity of myocarditis, which was accompanied by a reduction in heart weight to body weight ratio and heart weight. Captopril specifically interfered with cell-mediated immunity as myosin delayed-type hypersensitivity (DTH) was reduced, while anti-myosin Ab production was not affected. Captopril-treated, OVA-immunized mice also exhibited a decrease in OVA DTH. In myosin-immunized, untreated mice, injection of captopril directly into the test site also suppressed myosin DTH. Interestingly, captopril did not directly affect Ag-specific T cell responsiveness because neither in vivo nor in vitro captopril treatment affected the proliferation, IFN-γ secretion, or IL-2 secretion by Ag-stimulated cultured splenocytes. These results indicate that captopril ameliorates experimental autoimmune myocarditis and may act, at least in part, by interfering with the recruitment of cells to sites of inflammation and the local inflammatory environment.
α-Melanocyte-Stimulating Hormone Inhibits Allergic Airway Inflammation
Authors: Raap Ulrike , Brzoska Thomas , Sohl Sirius , Päth Günter , Emmel Jörg , Herz Udo , Braun Armin , Luger Thomas , Renz Harald
Published: April 2014

α-Melanocyte-stimulating hormone (α-MSH) is a neuropeptide controlling melanogenesis in pigmentary cells. In addition, its potent immunomodulatory and immunosuppressive activity has been recently described in cutaneous inflammatory disorders. Whether α-MSH is also produced in the lung and might play a role in the pathogenesis of inflammatory lung conditions, including allergic bronchial asthma, is unknown. Production and functional role of α-MSH were investigated in a murine model of allergic airway inflammation. α-MSH production was detected in bronchoalveolar lavage fluids. Although aerosol challenges stimulate α-MSH production in nonsensitized mice, this rapid and marked stimulation was absent in allergic animals. Treatment of allergic mice with α-MSH resulted in suppression of airway inflammation. These effects were mediated via IL-10 production, because IL-10 knockout mice were resistant to α-MSH treatment. This study provides evidence for a novel function of α-MSH linking neuroimmune functions in allergic airway inflammation.
Distinct Role of ZAP-70 and Src Homology 2 Domain-Containing Leukocyte Protein of 76 kDa in the Prolonged Activation of Extracellular Signal-Regulated Protein Kinase by the Stromal Cell-Derived Factor-1α/CXCL12 Chemokine
Authors: Kremer Kimberly N. , Humphreys Troy D. , Kumar Ashok , Qian Nan-Xin , Hedin Karen E.
Published: April 2014

Stimulation of T lymphocytes with the ligand for the CXCR4 chemokine receptor stromal cell-derived factor-1α (SDF-1α/CXCL12), results in prolonged activation of the extracellular signal-regulated kinases (ERK) ERK1 and ERK2. Because SDF-1α is unique among several chemokines in its ability to stimulate prolonged ERK activation, this pathway is thought to mediate special functions of SDF-1α that are not shared with other chemokines. However, the molecular mechanisms of this response are poorly understood. In this study we show that SDF-1α stimulation of prolonged ERK activation in Jurkat T cells requires both the ZAP-70 tyrosine kinase and the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) scaffold protein. This pathway involves ZAP-70-dependent tyrosine phosphorylation of SLP-76 at one or more of its tyrosines, 113, 128, and 145. Because TCR activates ERK via SLP-76-mediated activation of the linker of activated T cells (LAT) scaffold protein, we examined the role of LAT in SDF-1α-mediated ERK activation. However, neither the SLP-76 proline-rich domain that links to GADS and LAT, nor LAT, itself are required for SDF-1α to stimulate SLP-76 tyrosine phosphorylation or to activate ERK. Together, our results describe the distinct mechanism by which SDF-1α stimulates prolonged ERK activation in T cells and indicate that this pathway is specific for cells expressing both ZAP-70 and SLP-76.
Vulnerability of Human Neurons to T Cell-Mediated Cytotoxicity
Authors: Giuliani Fabrizio , Goodyer Cynthia G. , Antel Jack P. , Yong V. Wee
Published: April 2014

Axonal and neuronal loss occurs in inflammatory diseases of the CNS such as multiple sclerosis. The cause of the loss remains unclear. We report that polyclonally activated T cells align along axons and soma of cultured human neurons leading to substantial neuronal death. This occurs in an allogeneic and syngeneic manner in the absence of added Ag, requires T cells to be activated, and is mediated through cell contact-dependent mechanisms involving FasL, LFA-1, and CD40 but not MHC class I. Activated CD4+ and CD8+ T cell subsets are equally neuronal cytotoxic. In contrast to neurons, other CNS cell types (oligodendrocytes and astrocytes) are not killed by T cells. These results demonstrate for the first time the high and selective vulnerability of human neurons to T cells, and suggest that when enough activated T cells accumulate in the CNS, neuronal cytotoxicity can result through Ag-independent non-MHC class I mechanisms.
The Uterine NK Cell Population Requires IL-15 but These Cells Are Not Required for Pregnancy nor the Resolution of a Listeria monocytogenes Infection
Authors: Barber Ellen M. , Pollard Jeffrey W.
Published: April 2014

During pregnancy in mice, uterine natural killer (uNK) cells abundantly accumulate on the mesometrial side of the placenta. In this study, we show that the presence of both mature and immature uNK cells requires IL-15. Bone marrow transplantation of NK cell-negative mice due to null mutations in the recombination-activating gene (Rag) 2/common cytokine receptor γ-chain (Rag2−/−γc−/−) genes indicated that uNK cells originate from the bone marrow and require IL-15 to develop. NK cells are thought to be central players in the immune response to intracellular pathogens such as Listeria monocytogenes, a bacterium that also has a predilection for replication in the placenta. However, IL-15−/−, NK cell-deficient mice were relatively protected from this infection compared with wild-type mice, and during pregnancy the absence of NK cells did not compromise the immune response at this site. The loss of uNK cells results in decidual abnormalities, including thickening of the arterial walls with luminal narrowing and a hypocellular decidua basalis. These defects were rescued by bone marrow transplantation of the Rag2−/−γc−/− mice that restored the uNK cell population. The decidual abnormalities in the IL-15−/− mice however did not result in infertility as gestation times and litter sizes were comparable to those of wild-type mice. Fetal weights were mildly compromised, consistent with the arterial pathologies. These results show that uNK cells are not required for successful pregnancy and that NK cells are not essential for an adequate immune response to L. monocytogenes in either pregnant or nonpregnant mice.
Identification of Circulating Fibrocytes as Precursors of Bronchial Myofibroblasts in Asthma
Authors: Schmidt Matthias , Sun Guo , Stacey Martin A. , Mori Luca , Mattoli Sabrina
Published: April 2014

The mechanisms contributing to airway wall remodeling in asthma are under investigation to identify appropriate therapeutic targets. Bronchial myofibroblasts would represent an important target because they play a crucial role in the genesis of subepithelial fibrosis, a characteristic feature of the remodeling process, but their origin is poorly understood. We hypothesized that they originate from fibrocytes, circulating cells with the unique characteristic of expressing the hemopoietic stem cell Ag CD34 and collagen I. In this study we show that allergen exposure induces the accumulation of fibrocyte-like cells in the bronchial mucosa of patients with allergic asthma. These cells are CD34-positive; express collagen I and α-smooth muscle actin, a marker of myofibroblasts; and localize to areas of collagen deposition below the epithelium. By tracking labeled circulating fibrocytes in a mouse model of allergic asthma, we provide evidence that fibrocytes are indeed recruited into the bronchial tissue following allergen exposure and differentiate into myofibroblasts. We also show that human circulating fibrocytes acquire the myofibroblast phenotype under in vitro stimulation with fibrogenic cytokines that are produced in exaggerated quantities in asthmatic airways. These results indicate that circulating fibrocytes may function as myofibroblast precursors and may contribute to the genesis of subepithelial fibrosis in asthma.
Mast Cells in Airway Hyporesponsive C3H/HeJ Mice Express a Unique Isoform of the Signaling Protein Ras Guanine Nucleotide Releasing Protein 4 That Is Unresponsive to Diacylglycerol and Phorbol Esters
Authors: Li Lixin , Yang Yi , Wong Guang W. , Stevens Richard L.
Published: April 2014

cDNAs were recently isolated from BALB/c mouse mast cells (MCs) that encode the new signaling protein mouse Ras guanine nucleotide releasing protein 4 (mRasGRP4). The present study evaluates the expression pattern and biological activity of mRasGRP4 in a variety of mouse strains. As assessed immunohistochemically and by RNA analysis, mRasGRP4 is not coordinately expressed with any of its family members. Normally, mRasGRP4 is an MC-restricted protein in tissues, and kinetic studies revealed that mRasGRP4 is expressed relatively early in developing MCs. The expression of mRasGRP4 in the fetus before granulated MCs become abundant supports the conclusion that RasGRP4 participates in MC-specific differentiation pathways. Functional studies conducted with recombinant material revealed that mRasGRP4 is a cation-dependent, diacylglycerol (DAG)-regulated, guanine nucleotide exchange factor. Immunoelectron microscopic studies revealed that mRasGRP4 resides in either the cytosol or inner leaflet of the plasma membrane of the MC, implying that DAG controls the intracellular movement of this signaling protein in c-kit-stimulated MCs. The mRasGRP4 gene resides on chromosome 7B1 within a site that is prominently linked to baseline airway reactivity in backcrossed C3H/HeJ and A/J mice. A truncated isoform of mRasGRP4 that lacks its DAG-regulatory domain was isolated from C3H/HeJ mouse MCs. Sequence analysis showed that this isoform is the result of defective splicing of the precursor transcript. MCs play a central role in allergic inflammation. The discovery of a novel isoform of mRasGRP4 in hyporesponsive mice suggests that airway reactivity is influenced by RasGRP4-dependent signaling events in pulmonary MCs.
A Site on Laminin α5, AQARSAASKVKVSMKF, Induces Inflammatory Cell Production of Matrix Metalloproteinase-9 and Chemotaxis
Authors: Adair-Kirk Tracy L. , Atkinson Jeffrey J. , Broekelmann Thomas J. , Doi Masayuki , Tryggvason Karl , Miner Jeffrey H. , Mecham Robert P. , Senior Robert M.
Published: April 2014

Several peptide sequences in laminin α1, the α-chain of laminin (Ln)-1, mediate biological responses in vitro, but Ln-1 is rare in vivo. Since Ln-5 and Ln-10, which contain the α3 and α5 chains, respectively, are the most prominent laminin heterotrimers in normal adult tissues and few functional domains in other laminin chains have been identified, we are investigating the α3 and α5 chains for biological activities. Incubation of mouse macrophages with the laminin α5 peptide AQARSAASKVKVSMKF resulted in marked increase in matrix metalloproteinase (MMP)-9 mRNA and gelatinolytic activity in the conditioned media, whereas the corresponding α3 peptide QQARDAANKVAIPMRF had no effect. AQARSAASKVKVSMKF also induced expression of MMP-14, while MMP-2, MMP-3, MMP-7, MMP-12, and MMP-13 were not induced by this peptide. Deletion analyses indicated that a minimal sequence of ASKVKVSMKF was sufficient for increasing MMP-9 expression. AQARSAASKVKVSMKF was also chemotactic for neutrophils and macrophages in vitro, and induced accumulation of neutrophils and macrophages in lung airspaces in vivo following intranasal instillation into mice. Comparable accumulation occurred in MMP-9-deficient mice, indicating that MMP-9 was not required for AQARSAASKVKVSMKF-induced inflammatory cell emigration in the lung. A scrambled version of the minimal peptide, KAKSFVMVSK, was inactive. These data indicate that laminin α5-derived peptides can induce inflammatory cell chemotaxis and metalloproteinase activity.
Identification and Isolation of Dominant Susceptibility Loci for Pristane-Induced Arthritis
Authors: Olofsson Peter , Holmberg Jens , Pettersson Ulf , Holmdahl Rikard
Published: April 2014

Rheumatoid arthritis is a chronic inflammatory autoimmune disorder, controlled by multiple genes as well as environmental factors. With animal models, like the pristane-induced arthritis (PIA) in rats, it is possible to reduce the environmental effects and the genetic heterogeneity to identify chromosomal regions harboring genes responsible for the arthritis development. The PIA model has proved to be useful for identifying gene regions controlling different phases of the disease based on intercrosses between the resistant E3 and the susceptible DA rat. We have now performed a high-powered backcross analysis that confirms previous intercross-based data but also identifies additional loci. Earlier identified PIA loci were reproduced with high significance; Pia1 (MHC region on chromosome 20), Pia4 (chromosome 12), and Pia7 (chromosome 4) are all major regulators of PIA severity and were also found to operate in concert. These three loci were verified in congenic strains using both disease- and arthritis-inflammatory-related subphenotypes as traits. We were also able to detect five new quantitative trait loci with dominant effects on PIA: Pia10, Pia12, Pia13, Pia14, and Pia15 on chromosomes 10, 6, 7, 8, and 18, respectively. These data highlight the usefulness of the statistical power obtained in a backcross of a complex disease like arthritis.
Direct Binding of Toll-Like Receptor 2 to Zymosan, and Zymosan-Induced NF-κB Activation and TNF-α Secretion Are Down-Regulated by Lung Collectin Surfactant Protein A
Authors: Sato Morihito , Sano Hitomi , Iwaki Daisuke , Kudo Kazumi , Konishi Masanori , Takahashi Hiroki , Takahashi Toru , Imaizumi Hitoshi , Asai Yasufumi , Kuroki Yoshio
Published: April 2014

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-α secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-κB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-α secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.
Bcl6 Is a Transcriptional Repressor for theIL-18Gene
Authors: Takeda Nobue , Arima Masafumi , Tsuruoka Nobuhide , Okada Seiji , Hatano Masahiko , Sakamoto Akemi , Kohno Yoichi , Tokuhisa Takeshi
Published: April 2014

Bcl6 functions as a sequence-specific transcriptional repressor, and Bcl6-deficient (Bcl6−/−) mice have been reported to display Th2-type inflammatory diseases in multiple organs. Since IL-18 is a potent stimulator of Th2 cells, we examined the expression of IL-18 mRNA in bone marrow-derived macrophages from Bcl6−/− mice after LPS stimulation. Here we show that the expression was strikingly up-regulated after stimulation. The expression was also up-regulated in RAW264 cells, a murine macrophage cell line, by transfection with the dominant negative type of Bcl6 gene. We identified a putative Bcl6-binding DNA sequence (IL-18BS) upstream of exon 1 of the murine IL-18 gene and three IL-18BSs in the promoter region of human IL-18 gene. Binding of Bcl6 in nuclear protein from resting RAW264 cells to murine IL-18BS was detected by gel retardation assay and chromatin immunoprecipitation assay. The binding activity was diminished gradually in RAW264 cells after LPS stimulation. However, the amount of Bcl6 protein in these cells was constant over the period examined, suggesting the functional modification of Bcl6 protein after stimulation. Furthermore, murine IL-18BS was required for Bcl6 to repress the expression of the luciferase reporter gene under control of the IL-18 promoter. Taken together, Bcl6 is a key regulator of IL-18 production by macrophages.
Thymic Export Function and T Cell Homeostasis in Patients with Relapsing Remitting Multiple Sclerosis
Authors: Hug Andreas , Korporal Mirjam , Schröder Isabella , Haas Jürgen , Glatz Katharina , Storch-Hagenlocher Brigitte , Wildemann Brigitte
Published: April 2014

Multiple sclerosis (MS) is an inflammatory and possibly autoimmune mediated demyelinating disease of the CNS. Autoimmunity within the CNS may be triggered by dysfunction of peripheral immune tolerance mechanisms via changes in the homeostatic composition of peripheral T cells. We have assessed the release of naive T lymphocytes from the thymus in patients with relapsing remitting MS (RRMS) to identify alterations in the equilibrium of the peripheral T cell compartment. Thymic T cell production was estimated by measuring TCR excision circles (TRECs) as a traceable molecular marker in recent thymic emigrants. A total of 46 treatment-naive patients with active RRMS and 49 gender- and age-matched healthy persons were included in the study. The levels of TREC-expressing CD4+ and CD8+ T lymphocytes were significantly decreased in MS patients, and TREC quantities overall matched those of 30 years older healthy individuals. The average concentrations of TRECs/106 CD4+ and CD8+ T lymphocytes derived from MS patients and healthy donors were 26 × 103/106 and 28 × 103/106 vs 217 × 103/106 and 169 × 103/106, respectively. To account for any influence of T cell proliferation on TREC levels, we assayed T lymphocytes from additional patients with MS and normal individuals for telomere length (n = 20) and telomerase activity (8 MS patients, 16 controls), respectively. There were no significant differences between CD4+ and CD8+ T cells from MS patients and controls. Altogether, our findings suggest that an impaired thymic export function and, as a consequence, altered ability to maintain T cell homeostasis and immune tolerance may play an important pathogenic role in RRMS.
Persistent Infection of Human Microvascular Endothelial Cells by Coxsackie B Viruses Induces Increased Expression of Adhesion Molecules
Authors: Zanone Maria M. , Favaro Enrica , Conaldi Pier G. , Greening James , Bottelli Antonella , Perin Paolo Cavallo , Klein Nigel J. , Peakman Mark , Camussi Giovanni
Published: April 2014

Numerous studies indicate that enteroviruses, such as the Coxsackievirus (CV) group, are linked to autoimmune diseases. Virus tropism and tissue access are modulated by vascular endothelial cells (ECs), mainly at the level of the microvasculature. Data on the permissiveness of ECs to CV are, however, scanty and derived from studies on large vessel ECs. To examine the susceptibility of microvascular ECs to infection of group B CV (CVB), human dermal microvascular ECs (HMEC-1) were infected with three CVB strains, and the immunological phenotype of the infected cells was analyzed. All CVB persistently infected the EC cultures without producing overt cytopathic effects. Infected ECs retained endothelial characteristics. Release of infectious particles in cell supernatants persisted for up to 3 mo of culture. Infection up-regulated expression of the adhesion molecules ICAM-1 and VCAM-1, with the highest values detected during the first 30 days of infection (p < 0.05 vs uninfected HMEC-1). CVB infection increased production of the proinflammatory cytokines, IL-6, IL-8, and TNF-α, which may account for the enhanced expression of adhesion molecules. Parallel infection of macrovascular HUVEC had less evident effects on induction of ICAM-1 and did not significantly increase expression of VCAM-1. Moreover, mononuclear cell adhesion to CVB-infected HMEC-1 monolayers was increased, compared with uninfected monolayers. These results provide evidence that small vessel ECs can harbor a persistent viral infection, resulting in quantitative modification of adhesion molecule expression, which may contribute to the selective recruitment of subsets of leukocytes during inflammatory immune responses. Furthermore, our data confirm that the behavior against a viral challenge of ECs in large vessels and microvessels may differ.
Investigating the Impact of Helminth Products on Immune Responsiveness Using a TCR Transgenic Adoptive Transfer System
Authors: Boitelle Agnès , Scales Hannah E. , Di Lorenzo Caterina , Devaney Eileen , Kennedy Malcolm W. , Garside Paul , Lawrence Catherine E.
Published: April 2014

Helminth infections and their products have a potent immunomodulatory effect on the host immune system and can impair immune responses against unrelated Ags. In vitro studies have suggested that the immunomodulation by helminth extracts may be the result of bystander response bias toward a Th2 phenotype and/or an Ag-specific T lymphocyte proliferative hyporesponsiveness. The aim of this study was to determine the role of these potential mechanisms of immunosuppression in vivo. Therefore, using a sensitive model of CFSE-labeled OVA-specific TCR transgenic T lymphocyte adoptive transfer, we analyzed the effect of Ascaris suum body fluid (ABF) on the kinetics and amplitude of a primary OVA-specific T cell response as well as the Th1/Th2 profile of the response in wild-type and IL-4 knockout (KO) mice. We find that inhibition of delayed-type hypersensitivity by ABF was associated with a Th1/Th2 shift in wild-type animals, but not in IL-4 KO mice. The use of this model has allowed us to demonstrate that although the kinetics of the OVA-specific primary response was not affected by ABF, the expansion of the OVA-specific T lymphocytes was significantly inhibited in both wild-type and IL-4 KO mice. This inhibition was associated with a reduced proliferative capacity of these cells in vivo, distinct from anergy.
Selective Unresponsiveness to Conformational B Cell Epitopes of the Myelin Oligodendrocyte Glycoprotein in H-2b Mice
Authors: Bourquin Carole , Schubart Anna , Tobollik Stephanie , Mather Ian , Ogg Sherry , Liblau Roland , Linington Christopher
Published: April 2014

Autoantibodies directed against conformation-dependent epitopes of the extracellular domain of the myelin oligodendrocyte glycoprotein (MOGIgd) play a major role in the immunopathogenesis of demyelination in experimental autoimmune encephalomyelitis. We now demonstrate that one or more genes encoded within the MHC selectively censor the ability of H-2b mice to mount this conformation-dependent autoantibody response, while leaving T and B cell responses to linear MOGIgd epitopes intact. This novel form of selective B cell unresponsiveness discriminates between pathogenic and nonpathogenic Ab responses to MOG and determines whether or not Ab-dependent effector mechanisms play an important role in the pathogenesis of MOG-induced experimental autoimmune encephalomyelitis in the mouse.
Rat and Human Myelin Oligodendrocyte Glycoproteins Induce Experimental Autoimmune Encephalomyelitis by Different Mechanisms in C57BL/6 Mice
Authors: Oliver Alfred R. , Lyon Geoffrey M. , Ruddle Nancy H.
Published: April 2014

C57BL/6 mice immunized with the extracellular Ig-like domain of rat myelin oligodendrocyte glycoprotein (MOG) developed experimental autoimmune encephalomyelitis (EAE) resembling that induced by rodent MOG 35-55 in its B cell independence and predominantly mononuclear CNS infiltrate. In contrast, human MOG protein-induced EAE was B cell dependent with polymorphonuclear leukocytes. Human MOG differs from rat MOG at several residues, including a proline for serine substitution at position 42. Human MOG 35-55 was only weakly encephalitogenic, and a proline substitution in rat MOG at position 42 severely attenuated its encephalitogenicity. However, human MOG 35-55 was immunogenic, inducing proliferation and IFN-γ and IL-3 to human, but not rodent MOG 35-55. The B cell dependence of EAE induced by human MOG protein was not due to a requirement for Ag presentation by B cells, because spleen cells from B cell-deficient mice processed and presented human and rat MOG proteins to T cells. The different pathogenic mechanisms of human and rat MOG proteins might result from different Abs induced by these proteins. However, rat and human MOG proteins induced Abs to mouse MOG that were equivalent in titer and IgG subclass. These data demonstrate that EAE can be induced in C57BL/6 mice by two mechanisms, depending on the nature of the immunogen: an encephalitogenic T cell response to rat MOG or rodent MOG 35-55, or an encephalitogenic B cell response to epitopes on human MOG protein that most likely cross-react with mouse determinants.
More Stringent Conditions of Plasmid DNA Vaccination Are Required to Protect Grafted Versus Endogenous Islets in Nonobese Diabetic Mice
Authors: Seifarth Christian , Pop Shannon , Liu Bo , Wong Carmen P. , Tisch Roland
Published: April 2014

Recurrent autoimmune destruction of the insulin-producing β cells is a key factor limiting successful islet graft transplantation in type I diabetic patients. In this study, we investigated the feasibility of using an Ag-specific plasmid DNA (pDNA)-based strategy to protect pro-islets that had developed from a neonatal pancreas implanted under the kidney capsule of nonobese diabetic (NOD) mice. NOD recipient mice immunized with pDNA encoding a glutamic acid decarboxylase 65 (GAD65)-IgFc fusion protein (JwGAD65), IL-4 (JwIL4), and IL-10 (pIL10) exhibited an increased number of intact pro-islets expressing high levels of insulin 15 wk posttransplant, relative to NOD recipient mice immunized with pDNA encoding a hen egg lysozyme (HEL)-IgFc fusion protein (JwHEL)+JwIL4 and pIL10 or left untreated. Notably, the majority of grafted pro-islets detected in JwGAD65+JwIL4- plus pIL10-treated recipients was free of insulitis. In addition, administration of JwGAD65+JwIL4+pIL10 provided optimal protection for engrafted islets compared with recipient NOD mice treated with JwGAD65+JwIL4 or JwGAD65+pIL10, despite effective protection of endogenous islets mediated by the respective pDNA treatments. Efficient protection of pro-islet grafts correlated with a marked reduction in GAD65-specific IFN-γ reactivity and an increase in IL-10-secreting T cells. These results demonstrate that pDNA vaccination can be an effective strategy to mediate long-term protection of pro-islet grafts in an Ag-specific manner and that conditions are more stringent to suppress autoimmune destruction of grafted vs endogenous islets.
Relationships Among Murine CD11chigh Dendritic Cell Subsets as Revealed by Baseline Gene Expression Patterns
Authors: Edwards Alexander D. , Chaussabel Damien , Tomlinson Simon , Schulz Oliver , Sher Alan , Reis e Sousa Caetano
Published: April 2014

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11chigh DC subsets. CD4+, CD8α+, and CD8α− CD4− (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4+ and CD8α+ DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4+ DC, differing from them by <25 genes. This suggests that CD4+ and DN DC are closely related phylogenetically, whereas CD8α+ DC represent a more distant lineage, supporting the historical distinction between CD8α+ and CD8α− DC. However, staining patterns revealed that in contrast to CD4+ DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11chigh DC subsets that to date have been difficult to define as rigid distinctions.
Longitudinal Assessment of Changes in HIV-Specific Effector Activity in HIV-Infected Patients Starting Highly Active Antiretroviral Therapy in Primary Infection
Authors: Alter Galit , Hatzakis George , Tsoukas Christos Micheal , Pelley Karen , Rouleau Danielle , LeBlanc Roger , Baril Jean-Guy , Dion Harold , Lefebvre Eric , Thomas Réjean , Côté Pierre , Lapointe Normand , Routy Jean-Pierre , Sékaly Rafik-Pierre , Conway Brian , Bernard Nicole Flore
Published: April 2014

Both the magnitude and breadth of HIV-specific immunity were evaluated longitudinally on samples collected from six subjects starting highly active antiretroviral therapy (HAART) preseroconversion (group 1), 11 recently infected subjects starting HAART postseroconversion (group 2), five subjects starting HAART in the second half of the first year of infection (group 3), and six persons starting treatment in the chronic phase of infection (group 4). HIV-specific immunity was measured by IFN-γ ELISPOT, detecting the frequency of cells responding to a panel of HLA-restricted HIV-1 peptides. Intracellular cytokine staining was used to detect the frequency of HIV-1 Gag p55-specific CD4+ and CD8+ T cells in a subset of participants. The magnitude and breadth of HIV-specific responses persisted in all group 1 subjects and in 5 of 11 (45%) group 2 subjects. Both of these parameters declined in 6 of 11 (55%) group 2 and in all group 3 and 4 individuals. All persons who maintained detectable numbers of HIV-1 Gag p55-specific CD4+ and CD8+ T cells after starting HAART preserved the intensity and breadth of their HIV-specific effector response. Our results show that HIV-specific immunity can be preserved even if HAART is initiated beyond the acute phase of infection.
Short Term Administration of Costimulatory Blockade and Cyclophosphamide Induces Remission of Systemic Lupus Erythematosus Nephritis in NZB/W F1 Mice by a Mechanism Downstream of Renal Immune Complex Deposition
Authors: Schiffer Lena , Sinha Jayashree , Wang Xiaobo , Huang Weiqing , von Gonsdorff Gero , Schiffer Mario , Madaio Michael P. , Davidson Anne
Published: April 2014

NZB/W F1 mice with established nephritis were treated with a single dose of cyclophosphamide with or without a 2-wk course of murine CTLA4Ig, either alone or in combination with anti-CD154. Sixty to 80% of treated mice entered remission, and remission could be reinduced following relapse. A decrease in the frequency of anti-DNA-producing B cells and activated T cells was observed in treated mice, but this effect lasted only 3–6 wk, while remissions were sustained for up to 20 wk. Light microscopy of the kidneys of mice in remission revealed less glomerular inflammation, less tubular damage, and less infiltration of inflammatory cells. By immunofluorescence, however, IgG and C3 staining of glomeruli was no different in treated mice vs controls. Since chemokines and their receptors play an important role in inflammatory cell infiltration of affected organs in autoimmune diseases, we examined chemokine expression in the kidneys. Decreases in the expression of inflammatory cytokines and chemokines were evident in mice in the early stages of remission, but these differences were no longer present in late remission. Increased expression of CXCL13 was detected in the inflammatory infiltrates of the control NZB/NZW mice. Strikingly, we could not detect any CXCL13 in the kidneys of the treated group even in late remission. These findings suggest that costimulatory blockade together with cyclophosphamide influence the activation state of renal CD11c-positive cells and therefore lead to less B and T cell infiltration and nephritis.
Homeostasis of Naive and Memory CD4+ T Cells: IL-2 and IL-7 Differentially Regulate the Balance Between Proliferation and Fas-Mediated Apoptosis
Authors: Jaleco Sara , Swainson Louise , Dardalhon Valérie , Burjanadze Maryam , Kinet Sandrina , Taylor Naomi
Published: April 2014

Cytokines play a crucial role in the maintenance of polyclonal naive and memory T cell populations. It has previously been shown that ex vivo, the IL-7 cytokine induces the proliferation of naive recent thymic emigrants (RTE) isolated from umbilical cord blood but not mature adult-derived naive and memory human CD4+ T cells. We find that the combination of IL-2 and IL-7 strongly promotes the proliferation of RTE, whereas adult CD4+ T cells remain relatively unresponsive. Immunological activity is controlled by a balance between proliferation and apoptotic cell death. However, the relative contributions of IL-2 and IL-7 in regulating these processes in the absence of MHC/peptide signals are not known. Following exposure to either IL-2 or IL-7 alone, RTE, as well as mature naive and memory CD4+ T cells, are rendered only minimally sensitive to Fas-mediated cell death. However, in the presence of the two cytokines, Fas engagement results in a high level of caspase-dependent apoptosis in both RTE as well as naive adult CD4+ T cells. In contrast, equivalently treated memory CD4+ T cells are significantly less sensitive to Fas-induced cell death. The increased susceptibility of RTE and naive CD4+ T cells to Fas-induced apoptosis correlates with a significantly higher IL-2/IL-7-induced Fas expression on these T cell subsets than on memory CD4+ T cells. Thus, IL-2 and IL-7 regulate homeostasis by modulating the equilibrium between proliferation and apoptotic cell death in RTE and mature naive and memory T cell subsets.