Studies in rodents have implicated various cytokines as paracrine mediators of increased osteoclastogenesis during estrogen deficiency, but increases in RANKL, the final effector of osteoclastogenesis, have not been demonstrated. Thus, we isolated bone marrow mononuclear cells expressing RANKL on their surfaces by two-color flow cytometry using FITC-conjugated osteoprotegerin-Fc (OPG-Fc-FITC) as a probe. The cells were characterized as preosteoblastic marrow stromal cells (MSCs), T lymphocytes, or B lymphocytes by using Ab’s against bone alkaline phosphatase (BAP), CD3, and CD20, respectively, in 12 premenopausal women (Group A), 12 early postmenopausal women (Group B), and 12 age-matched, estrogen-treated postmenopausal women (Group C). Fluorescence intensity of OPG-Fc-FITC, an index of the surface concentration of RANKL per cell, was increased in Group B over Groups A and C by two- to threefold for MSCs, T cells, B cells, and total RANKL-expressing cells. Moreover, in the merged groups, RANKL expression per cell correlated directly with the bone resorption markers, serum C-terminal telopeptide of type I collagen and urine N-telopeptide of type I collagen, in all three cell types and inversely with serum 17β-estradiol for total RANKL-expressing cells. The data suggest that upregulation of RANKL on bone marrow cells is an important determinant of increased bone resorption induced by estrogen deficiency.
Guitty Eghbali-Fatourechi, Sundeep Khosla, Arunik Sanyal, William J. Boyle, David L. Lacey, B. Lawrence Riggs
Comparing lymphocyte responses to allergenic and nonallergenic foods could reveal the differences between pathogenic and normal immune responses to foods. Defining the cytokine-producing phenotypes of peanut-specific lymphocytes from peanut-allergic children, children who outgrew peanut allergy, and children who have always tolerated peanuts may be useful for understanding the mechanisms of food tolerance. Investigating immune responses against foods is hindered, however, by the fact that circulating food antigen–specific lymphocytes are very rare. In a novel approach we used carboxyfluorescein succinimidyl ester to detect peanut-specific lymphocytes by flow cytometry. We confirmed that these cells are indeed peanut specific by cloning. Peanut-allergic donors show Th2 polarization of cytokine production by peanut-specific cells (IFN-γ low, TNF-α low, IL-4 high, IL-5 high, IL-13 high). Conversely, nonallergic children and children who have outgrown their allergy show Th1 skewing to peanut antigens (IFN-γhigh, TNF-α high, IL-4 low, IL-5 low, IL-13low), similarly to nonallergenic food antigens (β-lactoglobulin, OVA). This finding suggests that peanut antigens do not intrinsically induce Th2 skewing, but that the type of response depends upon the donor’s allergic status. In conclusion, food allergic status is characterized by a Th2 response whereas Th1-skewed responses underlie oral tolerance.
Victor Turcanu, Soheila J. Maleki, Gideon Lack
Epidemiological studies from both iodine-sufficient and -deficient human populations strongly suggest that early maternal hypothyroxinemia (i.e., low circulating free thyroxine before onset of fetal thyroid function at midgestation) increases the risk of neurodevelopmental deficits of the fetus, whether or not the mother is clinically hypothyroid. Rat dams on a low iodine intake are hypothyroxinemic without being clinically hypothyroid because, as occurs in pregnant women, their circulating 3,5,3′-triiodothyronine level is usually normal. We studied cell migration and cytoarchitecture in the somatosensory cortex and hippocampus of the 40-day-old progeny of the iodine-deficient dams and found a significant proportion of cells at locations that were aberrant or inappropriate with respect to their birth date. Most of these cells were neurons, as assessed by single- and double-label immunostaining. The cytoarchitecture of the somatosensory cortex and hippocampus was also affected, layering was blurred, and, in the cortex, normal barrels were not formed. We believe that this is the first direct evidence of an alteration in fetal brain histogenesis and cytoarchitecture that could only be related to early maternal hypothyroxinemia. This condition may be 150–200 times more common than congenital hypothyroidism and ought to be prevented both by mass screening of free thyroxine in early pregnancy and by early iodine supplementation to avoid iodine deficiency, however mild.
Rosalía Lavado-Autric, Eva Ausó, José Victor García-Velasco, María del Carmen Arufe, Francisco Escobar del Rey, Pere Berbel, Gabriella Morreale de Escobar
The rapid and selective accumulation of neutrophils into the lungs is thought to underlie the pulmonary failure that leads to sepsis-related death. In this study we investigated whether neutrophil TLR4 is important in LPS-induced pulmonary neutrophil recruitment by creating chimeric mice (transferring bone marrow between TLR4+/+ and TLR4–/– mice). In TLR4+/+ mice receiving TLR4–/– bone marrow, 6 weeks after transplant TLR4 was absent in all circulating leukocytes as well as in resident macrophages (these mice were termed LeukocyteTLR4–/–), and these cells were completely nonresponsive to LPS. In TLR4–/– mice receiving TLR4+/+ bone marrow, endothelial cells but not leukocytes were deficient in TLR4 (EndotheliumTLR4–/–). Surprisingly, systemic LPS (0.5 mg/kg) induced a dramatic increase in neutrophil sequestration into the lungs of LeukocyteTLR4–/– mice over the first 4 hours. Concomitantly, numbers of circulating leukocytes decreased by 90%. By contrast, EndotheliumTLR4–/– mice showed very little increase in neutrophil sequestration in the lungs, suggesting that endothelium rather than leukocyte TLR4 was important. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte–endothelial cell interactions in LPS-treated EndotheliumTLR4–/– mice than in LPS-treated LeukocyteTLR4–/– mice. This is consistent with less sequestration of leukocytes into the lungs of EndotheliumTLR4–/– mice. In conclusion, our data challenge the view that LPS directly activates neutrophils to trap in lungs and suggest a far more important role than previously appreciated for the endothelial cells.
Graciela Andonegui, Claudine S. Bonder, Francis Green, Sarah C. Mullaly, Lori Zbytnuik, Eko Raharjo, Paul Kubes
The dissemination of IgA-dependent immunity between mucosal sites has important implications for mucosal immunoprotection and vaccine development. Epithelial cells in diverse gastrointestinal and nonintestinal mucosal tissues express the chemokine MEC/CCL28. Here we demonstrate that CCR10, a receptor for MEC, is selectively expressed by IgA Ab-secreting cells (large s/cIgA+CD38hiCD19int/–CD20–), including circulating IgA+ plasmablasts and almost all IgA+ plasma cells in the salivary gland, small intestine, large intestine, appendix, and tonsils. Few T cells in any mucosal tissue examined express CCR10. Moreover, tonsil IgA plasmablasts migrate to MEC, consistent with the selectivity of CCR10 expression. In contrast, CCR9, whose ligand TECK/CCL25 is predominantly restricted to the small intestine and thymus, is expressed by a fraction of IgA Ab-secreting cells and almost all T cells in the small intestine, but by only a small percentage of plasma cells and plasmablasts in other sites. These results point to a unifying role for CCR10 and its mucosal epithelial ligand MEC in the migration of circulating IgA plasmablasts and, together with other tissue-specific homing mechanisms, provides a mechanistic basis for the specific dissemination of IgA Ab-secreting cells after local immunization.
Eric J. Kunkel, Chang H. Kim, Nicole H. Lazarus, Mark A. Vierra, Dulce Soler, Edward P. Bowman, Eugene C. Butcher
Suppressor of cytokine signaling-1 (SOCS-1) is a negative regulator of cytokine signaling. To investigate the role of SOCS-1 in regulating inflammatory and immune responses in disease, acute inflammatory arthritis was induced in mice lacking SOCS-1. Expression of SOCS-1 protein was detected within synovial granulomas and pannus tissue of WT mice by day 7 following induction of acute arthritis. The severity of synovial inflammation and joint destruction at the peak of disease was greater in the absence of SOCS-1, although disease resolution occurred normally. There was an increased percentage of myeloid cells infiltrating the synovium in mice lacking SOCS-1, and SOCS-1 promoter activity was present in synovial macrophages, lymphocytes, and fibroblasts, but not granulocytes. The T cell response in draining LNs was also dysregulated, as popliteal LNs from mice lacking SOCS-1 contained approximately fivefold more cells at the peak of acute arthritis. These cells were hyperproliferative on exposure to antigen in vitro, and purified splenic CD4+ T cells from mice lacking SOCS-1 proliferated more strongly in response to stimulation with anti-CD3. Reporter gene expression was detected in CD4+ T cells bearing the activation markers CD25, CD44, and CD69. SOCS-1 is therefore expressed in hematopoietic and nonhematopoietic cell types in vivo and is an important regulator of acute inflammatory arthritis and of CD4+ T cell activation.
Paul J. Egan, Kate E. Lawlor, Warren S. Alexander, Ian P. Wicks
Accumulating evidence favors a role for proinsulin as a key autoantigen in diabetes. In the mouse, two proinsulin isoforms coexist. Most studies point to proinsulin 2 as the major isoform recognized by T cells in the NOD mouse. We studied mice in which a null proinsulin 2 mutation was transferred from proinsulin 2–deficient 129 mice onto the NOD background along with 16 genetic markers (including I-Ag7 MHC molecule) associated with diabetes. Intercross mice from the fourth backcross generation showed that proinsulin 2–/– mice develop accelerated insulitis and diabetes. The high prevalence of anti-insulin autoantibodies in proinsulin 2–/– mice indicates that diabetes acceleration relates to altered recognition of proinsulin. The prevalence of anti–glutamic acid decarboxylase autoantibodies and of sialitis is not increased in proinsulin 2–/– mice. We give evidence that proinsulin 2 expression leads to silencing of T cells specific for an epitope shared by proinsulin 1 and proinsulin 2. In the human, alleles located in the VNTR region flanking the insulin gene control β cell response to glucose and proinsulin expression in the thymus and are key determinants of diabetes susceptibility. Proinsulin 2–/– NOD mice provide a model to study the role of thymic expression of insulin in susceptibility to diabetes.
Karine Thébault-Baumont, Danielle Dubois-Laforgue, Patricia Krief, Jean-Paul Briand, Philippe Halbout, Karine Vallon-Geoffroy, Joëlle Morin, Véronique Laloux, Agnès Lehuen, Jean-Claude Carel, Jacques Jami, Sylviane Muller, Christian Boitard
FTY720 is a sphingosine-derived immunosuppressant. Phosphorylated FTY720 promotes T cell homing from spleen and peripheral blood to LNs by acting as an agonist for sphingosine-1-phosphate (S1P) receptors. Here we demonstrate that FTY720 enhances the activity of the sphingosine transporter Abcb1 (Mdr1) and the leukotriene C4 transporter Abcc1 (Mrp1). Both transporters must be active for FTY720-mediated T cell migration and LN homing. Migration and homing driven by FTY720, phosphorylated FTY720, or S1P also require 5-lipoxygenase–mediated synthesis of cysteinyl leukotrienes and their efflux from the cell. FTY720-mediated LN homing events further downstream are dependent on CCL19, CCL21, VLA-4α, and CD44. Use of T cells deficient in 5-lipoxygenase, Abcb1, and Abcc1, and comparison of the effects of FTY720 with those of S1P, suggest a model of sequential engagement of Abcb1, SP1 receptors, 5-lipoxygenase, and Abcc1 to enhance T cell migration and homing.
Shaun M. Honig, Shuang Fu, Xia Mao, Adam Yopp, Michael D. Gunn, Gwendalyn J. Randolph, Jonathan S. Bromberg
Our purpose here is to test the hypothesis that Randall’s plaques, calcium phosphate deposits in kidneys of patients with calcium renal stones, arise in unique anatomical regions of the kidney, their formation conditioned by specific stone-forming pathophysiologies. To test this hypothesis, we performed intraoperative biopsies of plaques in kidneys of idiopathic-calcium-stone formers and patients with stones due to obesity-related bypass procedures and obtained papillary specimens from non–stone formers after nephrectomy. Plaque originates in the basement membranes of the thin loops of Henle and spreads from there through the interstitium to beneath the urothelium. Patients who have undergone bypass surgery do not produce such plaque but instead form intratubular hydroxyapatite crystals in collecting ducts. Non–stone formers also do not form plaque. Plaque is specific to certain kinds of stone-forming patients and is initiated specifically in thin-limb basement membranes by mechanisms that remain to be elucidated.
Andrew P. Evan, James E. Lingeman, Fredric L. Coe, Joan H. Parks, Sharon B. Bledsoe, Youzhi Shao, Andre J. Sommer, Ryan F. Paterson, Ramsay L. Kuo, Marc Grynpas
We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR α subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the αV132L mutation. The analysis reveals that αV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the δ subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the β and ε subunits are without effect. The unique functional contribution of the α subunit cys-loop likely owes to its direct connection via a β strand to αW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.
Xin-Ming Shen, Kinji Ohno, Akira Tsujino, Joan M. Brengman, Monique Gingold, Steven M. Sine, Andrew G. Engel
Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl– secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K+ channels. We therefore speculated that a basolateral P2Y6 receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y6 receptor using crypt [Ca2+]i measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y6elevates cAMP, we coexpressed the P2Y1 or P2Y6 receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl– channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl– currents. In oocytes expressing the P2Y1 receptor, ATP transiently activated the endogenous Ca2+-activated Cl– current, but not CFTR. In contrast, in oocytes expressing the P2Y6receptor, UDP transiently activated the Ca2+-activated Cl– current and subsequently CFTR. CFTR Cl– currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y6 receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca2+]i and cAMP. In support of these data P2Y6 receptor stimulation differentially activates CFTR in Xenopus oocytes.
Michael Köttgen, Thomas Löffler, Christoph Jacobi, Roland Nitschke, Hermann Pavenstädt, Rainer Schreiber, Sebastian Frische, Søren Nielsen, Jens Leipziger
Prolactin is a peptide hormone produced by the anterior pituitary gland that is critical in lactation. Prolactin can also be produced by lymphocytes, and both B and T cells express prolactin receptors. These findings have suggested that prolactin has immunomodulatory functions. Studies in spontaneously autoimmune hosts have demonstrated a role for prolactin in augmenting autoreactivity. We chose to analyze prolactin effects on anti-DNA B cells in nonspontaneously autoimmune female BALB/c mice transgenic for the heavy chain of an anti-DNA antibody. Treatment with prolactin for 4 weeks induced a lupus-like phenotype with an increased number of transgene-expressing B cells, elevated serum anti-DNA antibody titers, and glomerular immunoglobulin deposits. Prolactin caused a decrease in the population of transitional B cells and an increase in mature follicular and marginal zone B cells. The DNA-reactive B cells had a follicular cell phenotype. Anti-DNA hybridomas demonstrated that prolactin alters selection of the naive B cell repertoire. The expansion and activation of anti-DNA B cells in prolactin-treated R4A-γ2b BALB/c mice was dependent on the presence of CD4+ T cells. Finally, treatment with prolactin was unable to break tolerance in R4A-γ2b transgenic C57Bl/6 mice, suggesting that responsiveness of the immune system to prolactin is genetically determined.
Elena Peeva, Daniel Michael, James Cleary, Jeffrey Rice, Xian Chen, Betty Diamond
Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of γ-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced γ-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.
Vladan P. Cokic, Reginald D. Smith, Bojana B. Beleslin-Cokic, Joyce M. Njoroge, Jeffery L. Miller, Mark T. Gladwin, Alan N. Schechter
Th2 cells are generated from naive CD4 T cells upon T cell receptor (TCR) recognition of antigen and IL-4 stimulation and play crucial roles in humoral immunity against infectious microorganisms and the pathogenesis of allergic and autoimmune diseases. A tyrosine phosphatase, SHP-1, that contains src homology 2 (SH2) domains is recognized as a negative regulator for various intracellular signaling molecules, including those downstream of the TCR and the IL-4 receptor. Here we assessed the role of SHP-1 in Th1/Th2 cell differentiation and in the development of Th2-dependent allergic airway inflammation by using a natural SHP-1 mutant, the motheaten mouse. CD4 T cells appear to develop normally in the heterozygous motheaten (me/+) thymus even though they express decreased amounts of SHP-1 (about one-third the level of wild-type thymus). The me/+ naive splenic CD4 T cells showed enhanced activation by IL-4 receptor–mediated signaling but only marginal enhancement of TCR-mediated signaling. Interestingly, the generation of Th2 cells was increased and specific cytokine production of mast cells was enhanced in me/+ mice. In an OVA-induced allergic airway inflammation model, eosinophilic inflammation, mucus hyperproduction, and airway hyperresponsiveness were enhanced in me/+ mice. Thus, SHP-1 may have a role as a negative regulator in the development of allergic responses, such as allergic asthma.
Tohru Kamata, Masakatsu Yamashita, Motoko Kimura, Kaoru Murata, Masamichi Inami, Chiori Shimizu, Kaoru Sugaya, Chrong-Reen Wang, Masaru Taniguchi, Toshinori Nakayama
Steroidal anti-inflammatory drugs induce proteins that inhibit phospholipase A2 (PLA2), including uteroglobin and lipocortin-1 (annexin I). Uteroglobin and lipocortin-1 retain several conserved sequences. Based on these sequences, several nonapeptides (antiflammins) were synthesized. These nonapeptides were shown to have anti-inflammatory effects in vitro and in vivo, possibly by inhibiting PLA2. Subsequent research showed that PLA2 is activated by transglutaminase 2 (TGase 2). We hypothesize here that TGase 2 inhibitors may increase the anti-inflammatory efficacy of inhibiting PLA2 activity. To test this theory, we constructed recombinant peptides containing sequences from pro-elafin (for inhibition of TGase 2), and from lipocortin-1, lipocortin-5, and uteroglobin (for inhibition of PLA2). The recombinant peptides, which had dual inhibitory effects on purified TGase 2 and PLA2, reversed the inflammation of allergic conjunctivitis to ragweed in a guinea pig model. The present work suggests that novel recombinant peptides may be safe and effective agents for the treatment of various inflammatory diseases.
Joonhong Sohn, Tae-Im Kim, Young-Hee Yoon, Joo-Yong Kim, Soo-Youl Kim
Acute liver failure caused by viral hepatitis or toxic damage involves both apoptotic and necrotic pathways. IGF binding protein-1 (IGFBP-1), a hepatocyte-derived secreted protein, is required for normal liver regeneration. To determine whether IGFBP-1 could prevent liver injury that entails direct stimulation of hepatocyte apoptosis, IGFBP-1–/– mice, IGFBP-1+/+ mice, and mice pretreated with Ab’s against IGFBP-1 were treated with a normally sublethal dose of Fas agonist. IGFBP-1 deficiency was associated with massive hepatocyte apoptosis and caspase activation within 3 hours of Fas agonist treatment, which could be corrected by pretreatment with IGFBP-1. IGFBP-1–deficient livers had enhanced signaling via the integrin receptor at early times (0.5 to 1 hour) after Fas agonist treatment accompanied by elevated activated matrix metalloproteinase-9 (MMP-9), a known target of fibronectin signaling and activator of TGF-β. Within 3 hours of Fas agonist treatment, elevated expression of active TGF-β1, a hepatocyte apoptogen, was observed in IGFBP-1–deficient livers that correlated with the appearance of the apoptotic process. Both MMP-9 and TGF-β1 expression were suppressed by IGFBP-1 treatment, supporting their role in the apoptotic process. IGFBP-1–/– mice also displayed increased injury in a toxic hepatic injury model caused by CCl4. These findings indicate that IGFBP-1 functions as a critical hepatic survival factor in the liver by reducing the level of proapoptotic signals.
Julia I. Leu, Mary Ann S. Crissey, Rebecca Taub
Using physiological, pharmacological, and gene disruption approaches, we demonstrate that proteinase-activated receptor-2 (PAR-2) plays a pivotal role in mediating chronic inflammation. Using an adjuvant monoarthritis model of chronic inflammation, joint swelling was substantially inhibited in PAR-2–deficient mice, being reduced by more than fourfold compared with wild-type mice, with virtually no histological evidence of joint damage. Mice heterozygous for PAR-2 gene disruption showed an intermediate phenotype. PAR-2 expression, normally limited to endothelial cells in small arterioles, was substantially upregulated 2 weeks after induction of inflammation, both in synovium and in other periarticular tissues. PAR-2 agonists showed potent proinflammatory effects as intra-articular injection of ASKH95, a novel synthetic PAR-2 agonist, induced prolonged joint swelling and synovial hyperemia. Given the absence of the chronic inflammatory response in the PAR-2–deficient mice, our findings demonstrate a key role for PAR-2 in mediating chronic inflammation, thereby identifying a novel and important therapeutic target for the management of chronic inflammatory diseases such as rheumatoid arthritis.
William R. Ferrell, John C. Lockhart, Elizabeth B. Kelso, Lynette Dunning, Robin Plevin, Stephen E. Meek, Andrew J.H. Smith, Gary D. Hunter, John S. McLean, Frances McGarry, Robert Ramage, Lu Jiang, Toru Kanke, Junichi Kawagoe
John A. Belperio, Michael P. Keane, Marie D. Burdick, Vedang Londhe, Ying Ying Xue, Kewang Li, Roderick J. Phillips, Robert M. Strieter
Dimitri Lodygin, Antje Menssen, Heiko Hermeking
Andrew P. Fontenot, Scott J. Canavera, Laia Gharavi, Lee S. Newman, Brian L. Kotzin
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