Cell, Vol. 116, 367–379, February 6, 2004, Copyright 2004 by Cell PressM Protein, a Classical Bacterial VirulenceDeterminant, Forms Complexes with Fibrinogenthat Induce Vascular Leakagemotactic signal, the PMNs become adherent and attachfirmly to the endothelium. The cells then start to extrava-sate into the infected tissue where they phagocytoseinvading bacteria. These adhesion processes involvethe sequential engagement of a number of different ad-Heiko Herwald,1,* Henning Cramer,1,5Matthias Mo¨rgelin,1Wayne Russell,1Ulla Sollenberg,2Anna Norrby-Teglund,3Hans Flodgaard,4Lennart Lindbom,2and Lars Bjo¨rck1hesion molecules both on PMNs and the endothelium,1Department of Cell and Molecular Biologyincluding selectins, integrins, and members of the familyLund Universityof intercellular adhesion molecules (ICAM) (for a review,Tornava¨gen 10see Ley, 2002).S-221 84 LundMembers of the ␤2integrin family (CD11a,b,c,d/CD18)Swedenare the most abundant integrins expressed by PMNs.2Department of Physiology and PharmacologyIn nonactivated PMNs, the majority of these integrins isKarolinska Institutetstored in intracellular granules, mainly in specific gran-S-171 77 Stockholmules and secretory vesicles, and only a minor portionSwedenis found attached to the plasma membrane (Dib and3Karolinska InstitutetAndersson, 2000). However, following stimulation spe-Center for Infectious Medicinecific granules and secretory vesicles may fuse with theHuddinge University Hospitalplasma membrane and induce an upregulation of the ␤2S-141 86 Stockholmintegrins at the cell surface. In resting cells, surface-Swedenexposed integrins remain in a low affinity state until they4Leukotech A/Sbecome activated by an “inside-out signaling” mecha-Symbion Science Parknism, resulting in a conformational change in the recep-Box 8tor and increased ligand binding. The number of ␤2integ-DK-2100 Copenhagenrin binding proteins is large and includes ligands thatDenmarkmediate firm adhesion to the endothelium, such as theICAMs, but also various plasma proteins including C3bi,factor X, and fibrinogen (for a review, see Crockett-SummaryTorabi, 1998). The interaction between fibrinogen and␤2integrins is regulated by divalent cations (Yan et al.,Increased vascular permeability is a key feature of1995), and whereas soluble fibrinogen binds only to acti-inflammatory conditions. In severe infections, leakagevated PMNs, immobilized fibrinogen can also interactof plasma from the vasculature induces a life-threaten-with unstimulated PMNs (Yan et al., 1995).ing hypotension. Streptococcus pyogenes, a major hu-Streptococcus pyogenes is a significant humanman bacterial pathogen, causes a toxic shock syn-pathogen giving rise to a wide spectrum of diseasesdrome (STSS) characterized by excessive plasmaranging from uncomplicated infections, such as pharyn-leakage and multi-organ failure. Here we find that Mgitis, impetigo, and erysipelas, to life-threatening condi-protein, released from the streptococcal surface,tions associated with shock and organ failure. S. pyo-forms complexes with fibrinogen, which by binding togenes expresses substantial amounts of M protein,␤2integrins of neutrophils, activate these cells. As a␣-helical coiled-coil surface proteins (for a review, seeresult, neutrophils release heparin binding protein, anFischetti, 1989), which represents one of the classicalinflammatory mediator inducing vascular leakage. Invirulence determinants of S. pyogenes promoting themice, injection of M protein or subcutaneous infectionsurvival of the bacterium in human blood (Lancefield,with S. pyogenes causes severe pulmonary damage1969). Apart from being associated with the bacterialcharacterized by leakage of plasma and blood cells.cell wall, M protein is also released from the surfaceThese lesions were prevented by treatment with a ␤2by the action of a cysteine proteinase secreted by theintegrin antagonist. In addition, M protein/fibrinogenbacteria (Berge and Bjo¨rck, 1995). Several reports havecomplexes were identified in tissue biopsies from ashown that M protein binds fibrinogen (Kantor, 1965)patient with necrotizing fasciitis and STSS, further un-with high affinity (A˚kesson et al., 1994). Here, we findderlining the pathogenic significance of such com-that M protein/fibrinogen complexes interact with ␤2in-plexes in severe streptococcal infections.tegrins and activate PMNs. This results in a massiveinflammatory response with profound pathophysiologi-Introductioncal consequences, which helps to explain the symptomsof streptococcal toxic shock syndrome (STSS), one ofPolymorphonuclear neutrophils (PMNs) are part of thethe most acute and severe forms of septic shock.first line of defense against bacterial infections. Underphysiological conditions nonactivated PMNs circulateResultsin the bloodstream. However, once activated by a che-Neutrophil Proteinases Release M1 Proteinfrom the Surface of S. pyogenes*Correspondence: [email protected] test whether M1 protein is released from the strepto-5Present address: Omnicare Clinical Research GmbH, D-65824Schwalbach/Ts., Germany.coccal surface following treatment with human neutro-Cell368Figure 1. Release of M1 Protein from theStreptococcal Surface Following Treatmentwith Supernatants from Stimulated PMNs(A) AP1 bacteria (2 ⫻ 109bacteria/ml) wereincubated with a serial dilution (100 ␮l, 10 ␮l,or 1 ␮l; lanes 2–4) of exudate from stimulatedPMNs (2 ⫻ 106cells/ml, see also ExperimentalProcedures) for 2 hr at 37⬚C. As a control,the bacteria were incubated with buffer alone(lane 1). Bacteria were centrifuged and theresulting supernatants were run on SDS-PAGE. Separated proteins were transferredonto nitrocellulose and probed with antibod-ies to M1 protein. Bound antibodies were de-tected by peroxidase-conjugated secondaryantibodies to rabbit immunoglobulin.(B) 10 ng purified M1 protein (lane 1), AP1surface proteins released with 100 ␮l neutro-phil secretion products (lane 2), and 10 ngpurified protein H (lane 3) were subjected toSDS-PAGE. After transfer onto nitrocellulosemembranes, the membranes were incubatedwith fibrinogen (2 ␮g/ml) followed by immu-nodetection with rabbit antibodies to humanfibrinogen and a peroxidase-conjugated sec-ondary antibody against rabbit immuno-globulin.(C) Transmission electron microscopy of thinsectioned AP1 bacteria before incubationwith exudate from stimulated PMNs.(D) AP1 bacteria after incubation with 2 ⫻ 108bacteria in