Published online December 1, 2006
PEDIATRICS Vol. 118 No. 6 December 2006, pp. 2498-2503 (doi:10.1542/10.1542/peds.2006-1845)

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ARTICLE

A Fast Procedure for the Detection of Defects in Toll-like Receptor Signaling

Horst von Bernuth, MD^a, Cheng-Lung Ku, MSc^a, Carlos Rodriguez-Gallego, MD^b, Shenying Zhang, MD^a, Ben-Zion Garty, MD^c, László Maródi, MD, PhD^d, Helen Chapel, MD^e, Maya Chrabieh^a, Richard L. Miller, PhD^f, Capucine Picard, MD, PhD^a,g, Anne Puel, PhD^a and Jean-Laurent Casanova, MD, PhD^a,h

^a Laboratory of Human Genetics of Infectious Diseases, University of Paris René Descartes, Institut National de la Santé et de la Recherche Médicale U550, Necker Medical School, Paris, France
^b Department of Immunology, Gran Canaria Dr Negrin Hospital, Las Palmas de Gran Canaria, Spain
^c Shneider Medical Center of Israel and Tel Aviv University Medical School, Tel Aviv, Israel
^d Department of Infectology and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
^e Department of Clinical Immunology, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom
^f 3M Center, St Paul, Minnesota
^g Immunodeficiency Study Center
^h Pediatric Hematology-Immunology Unit, Necker Hospital, Paris, France

OBJECTIVES. Inborn defects in Toll-like receptor signaling are recently described primary immunodeficiencies that predispose affected children to life-threatening infections. Patients with interleukin-1 receptor-associated kinase-4 deficiency are prone to invasive pneumococcal disease, and patients with UNC-93B deficiency are prone to herpes simplex virus encephalitis. These genetic disorders are underdiagnosed, partly because diagnosis currently requires expensive and time-consuming techniques available at only a few specialized centers worldwide. We, therefore, aimed to develop a cheap and fast test for the detection of defects in Toll-like receptor signaling.

PATIENTS AND METHODS. We used flow cytometry to evaluate the cleavage of membrane-bound L-selectin on granulocytes in 38 healthy controls and in 7 patients with genetically defined Toll-like receptor signaling defects (5 patients with interleukin-1 receptor-associated kinase-4 deficiency and 2 patients with UNC-93B deficiency), on activation with various Toll-like receptor agonists.

RESULTS. Impaired L-selectin shedding was observed with granulocytes from all of the interleukin-1 receptor-associated kinase-4-deficient patients on activation with agonists of Toll-like receptors 1/2, 2/6, 4, 7, and 8 and with granulocytes from all of the UNC-93B-deficient patients on activation with agonists of Toll-like receptors 7 and 8. All of the healthy controls responded to these stimuli.

CONCLUSIONS. The assessment of membrane-bound L-selectin cleavage on granulocytes by flow cytometry may prove useful for the detection of primary immunodeficiencies in the Toll-like receptor pathway, such as interleukin-1 receptor-associated kinase-4 deficiency and UNC-93B deficiency. This procedure is cheap and rapid. It may, therefore, be suitable for routine testing worldwide in children with invasive pneumococcal disease and in patients with herpes simplex encephalitis.

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Key Words: innate immunity • interleukin 1 receptor-associated kinase-4 deficiency • L-selectin • primary immunodeficiency • Toll-like receptors • UNC-93B deficiency

Abbreviations: TLR—Toll-like receptor • IRAK-4—interleukin-1-receptor-associated-kinase-4 • TNF-—tumor necrosis factor • IL—interleukin • LPS—lipopolysaccharide • ELISA—enzyme-linked immunosorbent assay • CD62L—L-selectin • PMA—phorbolmyristyl acetate • Pam₂CSK₄—synthetic diacylayed lipopeptide • Pam₃CSK₄—synthetic triacylayed lipopeptide • R-848—resiquimod • 3M-2—synthetic agonist of TLR8 • 3M-13—synthetic agonist of TLR7

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Accepted Aug 3, 2006.

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