Jussi Niemelä

CD73 is required for efficient entry of lymphocytes into the central nervous system during experimental autoimmune encephalomyelitis

CD73 is a cell surface enzyme of the purine catabolic pathway that catalyzes the breakdown of AMP to adenosine. Because of the strong immunosuppressive and antiinflammatory properties of adenosine, we predicted that cd73−/− mice would develop severe experimental autoimmune encephalomyelitis (EAE), an animal model for the central nervous system (CNS) inflammatory disease, multiple sclerosis. Surprisingly, cd73−/− mice were resistant to EAE. However, CD4 T cells from cd73−/− mice secreted more proinflammatory cytokines than wild-type (WT) mice and were able to induce EAE when transferred into naïve cd73+/+ T cell-deficient recipients. Therefore, the protection from EAE observed in cd73−/− mice was not caused by a deficiency in T cell responsiveness. Immunohistochemistry showed that cd73−/− mice had fewer infiltrating lymphocytes in their CNS compared with WT mice. Importantly, susceptibility to EAE could be induced in cd73−/− mice after the transfer of WT CD73+CD4+ T cells, suggesting that CD73 must be expressed either on T cells or in the CNS for disease induction. In the search for the source of CD73 in the CNS that might facilitate lymphocyte migration, immunohistochemistry revealed a lack of CD73 expression on brain endothelial cells and high expression in the choroid plexus epithelium which regulates lymphocyte immunosurveillance between the blood and cerebrospinal fluid. Because blockade of adenosine receptor signaling with the A2a adenosine receptor-specific antagonist SCH58261 protected WT mice from EAE induction, we conclude that CD73 expression and adenosine receptor signaling are required for the efficient entry of lymphocytes into the CNS during EAE development.

Altered purinergic signaling in CD73 deficient mice inhibits tumor progression

CD73/ecto‐5′‐nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti‐tumor immunity has not been studied. Here, we show that under physiological conditions CD73‐deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor‐infiltrating leukocytes there were fewer Tregs and mannose receptor‐positive macrophages, and increased IFN‐γ and NOS2 mRNA production in CD73‐deficient mice. Treatment of tumor‐bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor‐positive macrophages in the WT C57BL/6 mice but not in the CD73‐deficient mice. Pharmacological inhibition of CD73 with α,β‐methylene‐adenosine‐5′‐diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti‐tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.

IFN-α Induced Adenosine Production on the Endothelium: A Mechanism Mediated by CD73 (Ecto-5′-Nucleotidase) Up-Regulation

CD73 (ecto-5′-nucleotidase; EC 3.1.3.5) participates in lymphocyte binding to endothelial cells and converts extracellular AMP into a potent anti-inflammatory substance adenosine. However, the regulation of expression and function of CD73 has remained largely unknown. In this study, we show that IFN-α produces a time- and dose-dependent long-term up-regulation of CD73 on endothelial cells, but not on lymphocytes both at protein and RNA levels. Moreover, CD73-mediated production of adenosine is increased after IFN-α treatment on endothelial cells, resulting in a decrease in the permeability of these cells. Subsequent to induction with PMA, FMLP, dibutyryl cAMP, thrombin, histamine, IL-1β, TNF-α, and LPS, no marked changes in the level of CD73 expression on endothelial cells are observed. We also show that CD73 is up-regulated in vivo on the vasculature after intravesical treatment of urinary bladder cancers with IFN-α. In conclusion, distinct behavior of lymphocyte and endothelial CD73 subsequent to cytokine treatment further emphasizes the existence of cell type-specific mechanisms in the regulation of CD73 expression and function. Overall, these results suggest that IFN-α is a relevant in vivo regulator of CD73 in the endothelial-leukocyte microenvironment in infections/inflammations, and thus has a fundamental role in controlling the extent of inflammation via CD73-dependent adenosine production.

CD73 engagement promotes lymphocyte binding to endothelial cells via a lymphocyte function-associated antigen-1-dependent mechanism.

CD73 is a GPI-anchored lymphocyte adhesion molecule possessing an ecto-5′-nucleotidase enzyme activity. In this work, we show that engagement of lymphocyte CD73 increases lymphocyte binding to cultured endothelial cells (EC) in an LFA-1-dependent fashion. Engagement of CD73 by an anti-CD73 mAb 4G4 increases the adhesion of lymphocytes to cultured EC by about 80% compared with that of lymphocytes treated with a negative control Ab, and the increased adhesion can be blocked by an anti-CD18 mAb. The CD73-regulated increase in lymphocyte adhesion is not due to a conformational change leading to high-affinity LFA-1 receptors as assayed using mAb 24 against an activation-induced epitope of the molecule. Instead, CD73 engagement induces clustering of LFA-1 that is inhibitable by calpeptin, indicating involvement of Ca2+-dependent activation of a calpain-like enzyme in this process. In conclusion, the results shown here demonstrate that CD73 regulates the avidity of LFA-1 by clustering. This indicates a previously undescribed role for CD73 in controlling the poorly characterized activation step in the multistep cascade of lymphocyte extravasation. Moreover, these results suggest that in physiological conditions the activation step may result in clustering of LFA-1 rather than in an affinity change of the molecule.

IFN-β regulates CD73 and adenosine expression at the blood-brain barrier

IFN‐β treatment reduces the relapse rate in MS but its mechanism of action remains incompletely understood. Our aim was to clarify the beneficial effect of IFN‐β in the treatment of MS. We assessed the influence of IFN‐β treatment on (i) CD73 expression on the surface of primary cultures of human blood–brain barrier endothelial cells (BBB‐EC) and human astrocytes using immunofluorescence staining and flow cytometry, (ii) transmigration of CD4+ T lymphocytes using an in vitro model of BBB and (iii) CD73 enzyme activity, i.e. ecto‐5′‐nucleotidase activity in the serum of MS patients using a radiochemical assay. IFN‐β increases the expression of ecto‐5′‐nucleotidase both on BBB‐EC and astrocytes. As a consequence, lymphocyte transmigration through BBB‐EC is reduced. Importantly, this reduction can be reversed using α,β‐methyleneadenosine‐5′‐diphosphate, a specific inhibitor of ecto‐5′‐nucleotidase. CD73 is strongly expressed in microvasculature in samples of postmortem MS brain and, moreover, in the majority of MS patients there was a clear upregulation both in the soluble serum ecto‐5′‐nucleotidase activity and skin microvascular CD73 expression after IFN‐β treatment. Upregulation of ecto‐5′‐nucleotidase and a subsequent increase in adenosine production might contribute to the beneficial effects of IFN‐β on MS via enhancing the endothelial barrier function.

Mechanism of Action of IFN‐β in the Treatment of Multiple Sclerosis

Abstract:  IFN‐β treatment reduces the relapse rate in multiple sclerosis (MS), but the exact mechanism of action of the drug has remained elusive. CD73 (ecto‐5′‐nucleotidase) is an ectoenzyme, which produces adenosine from adenosine monophosphate (AMP) precursor by enzymatic dephosphorylation. AMP is known to be abundantly present at sites of inflammation, and more importantly adenosine, the product of CD73, is known to possess both anti‐inflammatory and neuroprotective activity. Our preliminary work has shown that IFN‐β increases the expression of ecto‐5′‐nucleotidase on endothelial cells (ECs) both in vitro and after systemic treatment of MS patients in vivo. In the majority of MS patients also an increase in the soluble serum CD73 was noted after IFN‐β treatment. Importantly, this correlated with the clinical outcome. CD73 expression on central nervous system (CNS) microvasculature was confirmed with stainings of frozen tissue sections of MS brain samples taken at autopsy. Adenosine, a known neuroprotective agent, might contribute to the beneficial effects of IFN‐β on MS.

Human rhinovirus C—Associated severe pneumonia in a neonate

Abstract We present a case of severe pneumonia, associated with a prolonged infection by a species C rhinovirus (HRV) in a 3-week old neonate. HRV RNA was identified in nasal and nasopharyngeal secretions, bronchoalveolar lavage and bronchial specimens, stool and urine, collected from the patient during a one-month period. No other viral or bacterial agents were detected. Sequence analysis of two regions of the viral genome, amplified directly from the clinical specimens revealed a novel HRV-C variant. These observations highlight the occurrence of severe neonatal infections caused by HRVs and the need of rapid viral diagnostics for their detection.

Identification of respiratory viruses with a novel point-of-care multianalyte antigen detection test in children with acute respiratory tract infection

Abstract Background Rapid etiological diagnosis of a respiratory virus infection may have impact on antiviral and antibiotic therapy, patient cohorting, and prediction of the clinical course. Most point-of-care tests for detection of respiratory viruses have limitations in diagnostic performance and clinical usability. A novel, multianalyte point-of-care antigen detection test system (mariPOC®; ArcDia International Oy Ltd., Turku, Finland) detects eight respiratory viruses (influenza A and B viruses, respiratory syncytial virus (RSV), adenovirus, human metapneumovirus, and parainfluenza type 1, 2, and 3 viruses) from a single nasopharyngeal swab specimen by a fully automated, random-access immunoassay method. Objectives To evaluate mariPOC® point-of-care test system in comparison with reverse transcription polymerase chain reaction (RT-PCR) in a pediatric emergency department setting. Study design Prospectively collected samples from 158 children (mean age, 1.8 years) with respiratory symptoms and/or fever were analyzed both by mariPOC® and by multiplex RT-PCR. Results The sensitivities and specificities (95% confidence intervals) of the mariPOC® test were for influenza A (n =7), 71% (38–100) and 100%; influenza B (n =22), 86% (72–100) and 98% (95–100); RSV (n =35), 89% (78–99) and 100%; adenovirus (n =12), 25% (1–50) and 97% (95–99); and for human metapneumovirus (n =8), 50% (15–85) and 100%, respectively. Parainfluenzaviruses were detected only in five patients. Conclusions This novel point-of-care test system is a rapid, practical, and specific method for simultaneous detection of eight respiratory viruses. Compared with RT-PCR, its sensitivity is moderately high for detection of RSV and influenza viruses, and low for adenovirus.

Accuracy and Feasibility of Point-Of-Care White Blood Cell Count and C-Reactive Protein Measurements at the Pediatric Emergency Department

Background Several point-of-care (POC) tests are available for evaluation of febrile patients, but the data about their performance in acute care setting is sparse. We investigated the analytical accuracy and feasibility of POC tests for white blood cell (WBC) count and C-reactive protein (CRP) at the pediatric emergency department (ED). Methods In the first part of the study, HemoCue WBC and Afinion AS100 CRP POC analyzers were compared with laboratory’s routine WBC (Sysmex XE-2100) and CRP (Modular P) analyzers in the hospital central laboratory in 77 and 48 clinical blood samples, respectively. The POC tests were then adopted in use at the pediatric ED. In the second part of the study, we compared WBC and CRP levels measured by POC and routine methods during 171 ED patient visits by 168 febrile children and adolescents. Attending physicians performed POC tests in capillary fingerprick samples. Results In parallel measurements in the laboratory both WBC and CRP POC analyzers showed good agreement with the reference methods. In febrile children at the emergency department (median age 2.4 years), physician performed POC determinations in capillary blood gave comparable results with those in venous blood analyzed in the laboratory. The mean difference between POC and reference test result was 1.1 E9/L (95% limits of agreement from -6.5 to 8.8 E9/L) for WBC and -1.2 mg/L (95% limits of agreement from -29.6 to 27.2 mg/L) for CRP. Conclusions POC tests are feasible and relatively accurate methods to assess CRP level and WBC count among febrile children at the ED.

Aetiology of febrile pharyngitis in children: Potential of myxovirus resistance protein A (MxA) as a biomarker of viral infection

Summary Objectives Besides group A streptococcus (GAS), microbial causes of pharyngitis in children are not well known. We aimed to document the viral and bacterial aetiology of pharyngitis and to assess the pathogenic role of viruses by determining the myxovirus resistance protein A (MxA) in the blood as a marker of interferon response. Methods In this prospective observational study, throat swabs and blood samples were collected from children (age 1–16 years) presenting to the emergency department with febrile pharyngitis. Microbial cause was sought by bacterial culture, polymerase chain reaction, and serology. Blood MxA level was determined. Results A potential pathogen was detected in 88% of 83 patients: GAS alone in 10%, GAS and viruses in 13%, group C or G streptococci alone in 2% and together with viruses in 3%, and viruses alone in 59% of cases. Enteroviruses, rhinoviruses, and adenoviruses were the most frequently detected viruses. Blood MxA levels were higher in children with viral (880 [245–1250] μg/L; median [IQR]) or concomitant GAS-viral (340 [150–710] μg/L) than in those with sole GAS (105 [80–160] μg/L) infections. Conclusions Detection of respiratory viruses simultaneously with elevated blood MxA levels supports the causative role of viruses in the majority of children with pharyngitis.