Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes

Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted ₂-integrin and urokinase receptor−mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2−3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented ₂-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.

As an immediate response towards bacterial infection or injury, leukocytes migrate from the blood stream into extravascular sites of inflammation. This coordinated sequence of adhesion and locomotion steps requires the expression and upregulation of various adhesion receptors on the surface of mobile and stationary vascular cells. Although leukocyte rolling depends on selectins, the firm adhesion to and transmigration through the endothelium is predominantly mediated by the ₂-integrins Mac-1 (CD11b/CD18, M2, CR3) and lymphocyte function-associated antigen-1 (LFA-1) (CD11/CD18, _L2) that interact with their major counter-receptor intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Mac-1 also regulates leukocyte adhesion to provisional matrix substrates including FBG, which is deposited at sites of inflammation and injury upon increased vascular permeability and damage^{23, 24}. Moreover, very late antigen-4 (VLA-4) (₄₁)-integrin mediates leukocyte adhesion to and transmigration through the endothelium by binding to endothelial vascular cell-adhesion molecule-1 (VCAM-1) and to FN in the extracellular matrix²⁵. Divalent cations, integrin-associated proteins as well as the urokinase plasminogen activator receptor (uPAR, CD87) interact with and thereby regulate integrin function. uPAR also directly mediates leukocyte adhesion to VN, a major adhesive component of the provisional wound-healing matrix, and thereby serves a dual role in pericellular proteolysis and as mediator of cellular contacts in a proteolysis-independent fashion. The VN−uPAR interaction is promoted by urokinase plasminogen activator (uPA) and blocked by plasminogen-activator inhibitor 1 (PAI-1), or two-chain high-molecular-weight kininogen (HKa)^{26, 27, 28}. Thus, adhesion-receptor crosstalk on leukocytes allows control of the strength and duration of adhesive reactions required for the spatio-temporal coordination of the host inflammatory response.

These aspects prompted us to investigate whether S. aureus Eap could influence the recruitment of leukocytes by binding to different extracellular and/or cell surface−associated adhesion proteins. Our results indicate that the secreted bacterial protein Eap specifically interacts not only with FBG, VN and FN, but more importantly with ICAM-1 on endothelial cells. By blocking ₂-integrin-dependent leukocyte adhesion to ICAM-1 and FBG, as well as uPAR-dependent leukocyte adhesion to VN, Eap inhibits the recruitment of neutrophils into the inflamed peritoneum in vivo. These data indicate a novel function of Eap as a prominent anti-inflammatory factor with possible therapeutic use.

The adhesion of differentiated myelo-monocytic U937 cells to immobilized FBG is predominantly mediated by the ₂-integrin Mac-1, whereas adhesion to ICAM-1 on endothelial cells is mediated by Mac-1 and LFA-1. Adhesion to FN is mediated by the ₁-integrin VLA-4. All these adhesion reactions are upregulated by Mn²⁺ or phorbol ester (PMA). Moreover, leukocyte adhesion to immobilized VN is mediated by uPAR, whereas uPA augments this adhesion by increasing the affinity of the uPAR−VN interaction^{29, 30, 31}. In order to characterize the role of S. aureus Eap from three strains, they were included in adhesion of U937 cells to different substrates. VLA-4-dependent U937 cell adhesion to FN was not affected by any of the Eap forms (Fig. 1a), as Eap did not interfere with the interaction between FN and VLA-4 (data not shown). However, all three Eap forms dose-dependently inhibited the uPAR-dependent adhesion to VN both in the absence of uPA as well as under uPA stimulation (Fig. 1b). Moreover, Mac-1-mediated monocyte adhesion to FBG (Fig. 1c) and Mac-1/LFA-1-dependent monocyte adhesion to immobilized ICAM-1 (Fig. 1d) were completely blocked by all three forms of Eap, both under basal conditions or under PMA or Mn²⁺-stimulation. As expected, Eap could also abolish the Mac-1/LFA-1-mediated adhesion of monocytes to an endothelial cell monolayer (data not shown). The inhibition by Eap was comparable or even stronger than the effects of previously described inhibitors of uPAR or ₂-integrin dependent cell adhesion, such as PAI-1 or HKa (Table 1)^{27, 32}. In contrast to Eap, S. aureus clumping factor did not affect any of the above described adhesion reactions. Finally, immobilized Eap itself did not present any cell-adhesive properties in these assays (data not shown).

Figure 1. Influence of Eap on the adhesion of myelomonocytic U937 cells. a−c, U937 cell adhesion to immobilized FN with PMA (50 ng/ml) (a), immobilized VN with uPA (50 nM) (b) and to immobilized FBG with PMA (50 ng/ml) (c) was carried out in the absence or presence of Eap N (), EapW () or Eap7 (). d, U937 cell adhesion to immobilized ICAM-1 was analyzed in the absence () or presence of Mn2+ (100 M) () or PMA (50 ng/ml) () without (-) or together with EapN, EapW, Eap7 or clumping factor (CLF) (each 5 g/ml) as indicated. Cell adhesion is expressed as absorbance at 590 nm and data are mean s.e.m. (n = 3) of a typical experiment. Similar results were obtained in at least 3 separate experiments; *: P < 0.01 versus control. Full Figure and legend (34K)

Table 1. Anti-adhesive effect of Eap on U937 cells Full Table

Figure 2. Interference of Eap with adhesion receptors of leukocytes. a and b, Binding of FBG (2 g/ml) (a) to immobilized Mac-1 (5 g/ml) and of ICAM-1 (10 g/ml) (b) to immobilized Mac-1 or LFA-1 (each 5 g/ml) was studied in the absence (-) or presence of EDTA (10 mM), EapN, EapW or Eap7 (each 5 g/ml) as indicated. c, Binding of VN (2 g/ml) to immobilized suPAR (5 g/ml) was studied without () or together with 50 nM uPA () in the absence (-) or presence of EapN, EapW, Eap7 (each 5 g/ml) or PAI-1 (100 nM) as indicated. Specific binding expressed as absorbance at 405 nm and data are mean s.e.m. (n = 3) of a typical experiment. Similar results were obtained in at least 3 separate experiments. *, P < 0.05 versus control; **, P < 0.01 versus control. Full Figure and legend (58K)

Figure 3. Binding of Eap to different adhesion receptors and extracellular matrix proteins. Binding of Eap7 (2 g/ml) to immobilized suPAR, Mac-1, LFA-1, ICAM-1 (5 g/ml each), FN, FBG or VN (4 g/ml each) was studied in the absence () or presence of heparin (10 g/ml) (). Specific binding is expressed as absorbance at 405 nm, and data are mean s.e.m. (n = 3) of a typical experiment. Similar results were obtained in 3 separate experiments; *, P < 0.01. Full Figure and legend (21K)

Figure 4. Contribution of Eap in the adhesion of S. aureus to ICAM-1. a, Adhesion of S. aureus strain Newman () and the Eap-deficient strain AH12 () to immobilized FBG, FN, or ICAM-1 (each 5 g/ml) was analyzed. Number of adherent bacteria is expressed as percent of total added bacteria, and data are mean s.e.m. (n = 3) of a typical experiment. Similar results were obtained in at least 3 separate experiments; **, P < 0.005; n.s., not significant. b, The adhesion of S. aureus strain Newman () and the Eap-deficient strain AH12 () to immobilized ICAM-1 was studied in the absence or presence of increasing concentrations of EapN. Number of adherent bacteria is expressed as percent of total added bacteria, and data are mean s.e.m. (n = 3) of a typical experiment. Similar results were obtained in at least 3 separate experiments; *, P < 0.05 versus control; **, P < 0.005 versus control. Full Figure and legend (27K)

The interactions described above indicate that Eap blocks leukocyte adhesion, a process that is pivotal for the recruitment of inflammatory cells to the infected tissue. To prove that Eap indeed regulates such processes, we tested the effect of Eap in vivo in an acute mouse model of inflammation. Peritonitis was induced by thioglycollate injection, and after four hours there was an expected increase in the total leukocyte count, mostly attributable to emigrated neutrophils. The percentage of neutrophils among all leukocytes after 4 hours was 50−60% as compared with 3−10% 1h after stimulation^{33, 34}. The use of blocking antibodies against LFA-1, Mac-1 or ICAM-1 30 minutes before the induction of peritonitis resulted in a 50−75% inhibition of neutrophil extravasation into the inflamed peritoneum at 4 hours after thioglycollate injection (Fig. 5a and b); isotype-matched control antibody had no effect at all (data not shown). At one and four hours after thioglycollate injection, neutrophil recruitment to the peritoneum was significantly reduced in mice pretreated intravenously with Eap7 (50, 75 and 100 g per mouse). The maximal inhibition (>75%) was obtained at 4 hours with 100 g Eap (Fig. 5a). Similarly, Eap injected intraperitoneally also blocked neutrophil recruitment by 50−60% (Fig. 5b).

Figure 5. Inhibition of neutrophil emigration by Eap in acute inflammation in vivo. a and b, After thioglycollate injection into the mouse peritoneum to induce acute inflammation, the number of neutrophils in the peritoneal lavage at 1 h and 4 h was analyzed. a, Before thioglycollate administration, mice were treated by intravenous injection with PBS (), with a blocking mAb against mouse -subunit of LFA-1 (), with a blocking mAb against mouse -subunit of Mac-1 () (each 100 g) or with Eap7 (100 g, ). b, Before thioglycollate administration, mice were treated by intraperitoneal injection with PBS (), or Eap7 (), or by intravenous injection with a blocking mAb against mouse ICAM-1 (). Data are mean s.e.m. (n = 4 mice per treatment) of a typical experiment. Similar results were obtained in three separate sets of experiments; *: P < 0.001 as compared with control (PBS). (c-d) Following S. aureus injection into the mouse peritoneum to induce acute inflammation, the number of neutrophils in the peritoneal lavage at 1 h and 5 h was determined. c, Mice received intraperitoneally 1 ml each of chemically defined medium HHW41 (-; ), strain Newman or Eap-deficient strain AH12 as indicated. Both strains (1 109 c.f.u. each) were injected together with their 15 h conditioned medium (), or were washed after the 15 h incubation period, resuspended in PBS and injected together with this buffer (). d, 30 min before the intraperitoneal administration of S. aureus (1 109 c.f.u. of either strain Newman or strain AH12), mice were treated intravenously with PBS () or with a blocking mAb against mouse ICAM-1 (100 g, ). Data are mean s.e.m. (n = 4 mice per treatment) of a typical experiment. Similar results were obtained in 2 separate sets of experiments; *, P < 0.01. Full Figure and legend (29K)

To further examine the anti-inflammatory role of Eap in vivo, peritonitis was induced by intraperitoneal injection of the S. aureus strain Newman and strain AH12 into different mouse groups. As Eap is partially secreted from S. aureus and partially rebinds to the bacterial surface, two different protocols were used. First, strains Newman and AH12 were cultivated for 6 and 15 hours and were injected intraperitoneally together with their 6- or 15-hour conditioned medium (chemically defined medium, HHW). Second, both strains were washed after the 6- and the 15-hour incubation period, resuspended in PBS and then injected. In both settings, there was a marked difference in the outcome of neutrophil recruitment between Eap-positive and -deficient strains. Mice injected with strain AH12 had a 2−3-fold higher number of neutrophils emigrating into the peritoneum as compared with mice injected with strain Newman. This difference was lower but still significant when the experiment was performed with washed bacteria (that is, when the Eap-rich 6-h or 15-h conditioned medium was removed) (Fig. 5c; data for 6-h incubated bacteria not shown). For comparison, peritonitis induced by isolated chemically defined medium HHW was found to be negligible (Fig. 5c). Neutrophil emigration in response to intraperitoneal injection of both strain Newman and strain AH12 was blocked by antibody against ICAM-1, indicating that there was no difference in the mechanism of neutrophil recruitment between mice that received either strain (Fig. 5d). Thus, Eap inhibits ₂-integrin-dependent neutrophil emigration in vivo.

In a system using purified components, Eap bound to proteins of the extracellular matrix such as VN, FBG and FN, and a novel binding of Eap to ICAM-1 was described. These interactions inhibited the association of Mac-1 with ICAM-1 or FBG, the binding of LFA-1 to ICAM-1 as well as the binding of VN to uPAR. In contrast, Eap did not affect the interaction between FN and its receptor VLA-4. These observations were consequently extended by our findings that Eap blocked the respective functional leukocyte adhesion systems. Finally, in vivo experiments demonstrated a potent inhibitory capacity of isolated Eap on ₂-integrin-dependent neutrophil recruitment into the inflamed peritoneum in mice. The concentrations of Eap used in our assays are similar to those found in the supernatant of S. aureus strains²². The anti-inflammatory role of Eap was further corroborated by the marked difference in neutrophil recruitment during peritonitis induced by Eap-positive compared with Eap-deficient S. aureus strains; neutrophil emigration into the inflamed peritoneum was 2−3-fold higher in mice that received strain AH12. This difference was reduced but still significant when peritonitis was studied with bacteria that had been separated from any additional Eap present in the conditioned medium. Thus, the anti-inflammatory activity can be attributed not only to Eap in the conditioned medium, but also to Eap that may be produced and released in vivo. Thus, Eap potently blocks ICAM-1-dependent leukocyte−endothelium interactions and the subsequent acute inflammatory response against S. aureus

	Top

Reagents were provided from these sources: isolated Mac-1, LFA-1 and ICAM-1 (from S. Bodary), insect cell-derived recombinant soluble uPAR (suPAR) (from D.B. Cines), PAI-1 (from P. Declerck), blocking monoclonal antibody (mAb) against human CD18, 60.3 (from J. Harlan), mAb against uPAR (ref. 37) (G. Hoyer-Hansen), mAb against ICAM-1, FBG and FN (DAKO, Hamburg, Germany), MnCl₂, FBG and FN (Sigma, Munich, Germany), vitamin D3 (Biomol, Hamburg, Germany), transforming growth factor- (R&D Systems, Boston, Massachusetts), uPA (Medac, Hamburg, Germany), 1- and 2-chain HKa (Enzyme Research Laboratories, South Bend, Indiana) and blocking mAbs against mouse Mac-1 (M1/70), mouse LFA-1 (M17/4) and mouse ICAM-1 (3E2), which were used for in vivo inhibition studies (Pharmingen, Hamburg, Germany). VN was purified from human plasma and converted to the multimeric form³⁸. S. aureus clumping factor and polyclonal antibodies directed against recombinant EapN were described^{39, 40}.

Myelo-monocytic U937 cells and human umbilical vein endothelial cells (HUVEC) were cultivated as described²⁹. Adhesion of U937 cells to immobilized VN, FN, ICAM-1, FBG or Eap (5 g/ml each) (and to BSA as control), as well as adhesion of U937 cells to cultured HUVECs was tested as described^{29, 30, 31, 32}.

Binding of FBG to immobilized Mac-1, of ICAM-1 to each immobilized Mac-1 or LFA-1 and of VN to immobilized uPAR was performed^{29, 32}. Alternatively, Eap (2 g/ml) binding to immobilized uPAR, Mac-1, LFA-1, ICAM-1, VN, FBG, FN or BSA (each 5 g/ml) or the binding of VN, FBG, FN or ICAM-1 (2 g/ml each) to immobilized Eap (5 g/ml) was performed in TBS containing 0.3% BSA, 0.05% Tween-20. Following incubation for 2h at 22 °C in each case, the respective anti-ligand antibody was added, followed by addition of appropriate secondary peroxidase-conjugated antibody (DAKO) and the substrate ABTS and binding was quantified at 405 nm. Nonspecific binding to BSA-coated wells was used as blank and was subtracted to calculate specific binding.

Polystyrene microtiter plates were coated with FBG, FN or ICAM-1 (5 g/ml each), dissolved in bicarbonate buffer, pH 9.6 and blocked with 3% BSA. S. aureus strain Newman or Eap-deficient strain AH12, each labeled with 1 M 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF AM) (Molecular Probes, Göttingen, Germany), were added at a density of 1 10⁶ cells per well and incubated for 1 h at 37 °C. Following a washing step, bacterial adhesion was quantified (as percentage of total cells added) in a fluorescence microplate reader (Bio-Tek, Neufahrn, Germany).

Thioglycollate-induced peritonitis in NMRI mice (Charles River Wiga, Sulzfeld, Germany) was performed as described^{32, 33, 34}. For inhibition studies, 30 min before the injection of thioglycollate, the following compounds were administered intravenously: 1) 100 g mAb against mouse Mac-1 or mouse LFA-1; 2) 100 g mAb against mouse ICAM-1 in PBS; or 3) 50−100 g of Eap in PBS. In a parallel group, mice received 100 g Eap intraperitoneally. Control mice were treated with the same volume of PBS or isotype-matched control antibodies. At 1 and 4 h following injection of thioglycollate mice were killed, the peritoneal lavage was collected and the number of emigrated neutrophils was quantified³². For the induction of bacterial peritonitis⁴², mice received 1 10⁹ c.f.u. of S. aureus Eap-positive strain Newman or Eap-deficient strain AH12 in a final volume of 1 ml intraperitoneally. First, bacterial strains were preincubated in chemically defined medium⁴¹ for 6 or 15 h followed by injection together with their 6- or 15-h conditioned medium. Second, another portion of the 6- or 15-h incubated bacteria (Newman or AH12) was washed twice in PBS and were injected in PBS. 5 h after the injection of bacteria, the number of emigrated neutrophils was quantified³². Animal experiments were approved by the local ethics committee.

For the wound-infection model, NMRI mice were anaesthetized and a 5-7 mm cut was made through the skin in the back. S. aureus strain Newman or AH12 (100 l, 3 10⁷ c.f.u.) was applied to separate wounds, giving 90% infection rate (as determined in pilot experiments). Wounds were sealed with sterile clips, and the animals were killed 4 d later. Abscess sizes were determined on a scale from 0 to +++, and after excision, they were homogenized and the c.f.u. were determined. Mice were also subjected to intravenous injections of 200 l of either strain Newman or AH12 (4 10⁶ c.f.u.) and 5 d later bacterial counts in the homogenized kidneys were determined.

	Top

1. Lowy, F.D. Staphylococcus aureus infections. N. Engl. J. Med. 339, 520–532 (1998). | Article | PubMed  | ISI | ChemPort |
2. Brumfitt, W. & Hamilton-Miller, J. Methicillin-resistant Staphylococcus aureus. N. Engl. J. Med. 320, 1188–1196 (1989). | PubMed  | ISI | ChemPort |
3. Waldvogel, F.A. New resistance in Staphylococcus aureus. N. Engl. J. Med. 340, 556–557 (1999). | Article | PubMed  | ISI | ChemPort |
4. Schlievert, P.M. Role of superantigens in human disease. J. Infect. Dis. 167, 997–1002 (1993). | PubMed  | ISI | ChemPort |
5. Kim, J., Urban, R.G., Strominger, J.L. & Wiley, D.C. Toxic shock syndrome toxin-1 complexed with a class II major histocompatibility molecule HLA-DR1. Science 266, 1870–1874 (1994). | PubMed  | ISI | ChemPort |
6. Jardetzky, T.S. et al. Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen. Nature 368, 711–718 (1994). | Article | PubMed  | ISI | ChemPort |
7. Bodén, M. & Flock, J.I. Fibrinogen-binding protein/clumping factor from Staphylococcus aureus. Infect. Immun. 57, 2358–2363 (1989). | PubMed  | ISI |
8. Flock, J.I. et al. Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus. EMBO J. 6, 2351–2357 (1987). | PubMed  | ISI | ChemPort |
9. McDevitt, D., Francois, P., Vaudaux, P. & Foster, T.J. Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol. Microbiol. 11, 237–248 (1994). | PubMed  | ISI | ChemPort |
10. Park, P.W., Rosenbloom, J., Abrams, W.R., Rosenbloom, J. & Mecham, R.P. Molecular cloning and expression of the gene for elastin binding protein (ebpS) in Staphylococcus aureus. J. Biol. Chem. 271, 15803–15809 (1996). | Article | PubMed  | ISI | ChemPort |
11. Patti, J.M. et al. Molecular characterization and expression of a gene encoding a Staphylococcus aureus collagen adhesin. J. Biol. Chem. 267, 4766–4772 (1992). | PubMed  | ISI | ChemPort |
12. Paulsson, M., Liang, O., Ascencio, F. & Wadström, T. Vitronectin binding surface proteins of Staphylococcus aureus. Zentbl. Bakteriol. 277, 54–64 (1992). | ChemPort |
13. Flock, J.I. Extracellular-matrix-binding proteins as targets for the prevention of Staphylococcus aureus infections. Mol. Med. Today 12, 532–537 (1999). | Article |
14. Moreillon, P. et al. Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis. Infect. Immun. 63, 4738–4743 (1995). | PubMed  | ISI | ChemPort |
15. Sinha, B. et al. Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells. Infect. Immun. 68, 6871–6878 (2000). | Article | PubMed  | ISI | ChemPort |
16. Sinha, B. et al. Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin 51. Cell. Microbiol. 1, 101–117 (1999). | Article | PubMed  | ISI | ChemPort |
17. Hendrix, H., Lindhout, T., Mertens, K., Engels, W. & Hemker, H.C. Activation of human prothrombin by stoichiometric levels of staphylocoagulase. J. Biol. Chem. 258, 3637–3644 (1983). | PubMed  | ISI | ChemPort |
18. Sawai, T. et al. Role of coagulase in a murine model of hematogenous pulmonary infection induced by intravenous injection of Staphylococcus aureus enmeshed in agar beads. Infect. Immun. 65, 466–471 (1997). | PubMed  | ISI | ChemPort |
19. Palma, M., Nozohoor, S., Schenning, T., Heimdahl, A. & Flock, J.I. Lack of the extracellular 19-kilodalton fibrinogen-binding protein from Staphylococcus aureus decreases virulence in experimental wound infection. Infect. Immun. 64, 5284–5289 (1996). | PubMed  | ISI | ChemPort |
20. Jönsson, K., McDevitt, D., McGavin, M.H., Patti, J.M. & Höök, M. Staphylococcus aureus expresses a major histocompatibility complex class II analog. J. Biol. Chem. 270, 21457–21460 (1995). | Article | PubMed  | ISI |
21. McGavin, M.H., Krajewska-Pietrasik, D., Rydén, C. & Höök, M. Identification of a Staphylococcus aureus extracellular matrix-binding protein with broad specificity. Infect. Immun. 61, 2479–2485 (1993). | PubMed  | ISI | ChemPort |
22. Palma, M., Haggar, A., Flock, J.I. Adherence of Staphylococcus aureus is enhanced by an endogenous secreted protein with broad binding activity. J. Bacteriol. 181, 2840–2845 (1999). | PubMed  | ISI | ChemPort |
23. Springer, T.A. Traffic signals for lymphocyte recirculation and leukocyte emigration: The multistep paradigm. Cell 76, 301–314 (1994). | Article | PubMed  | ISI | ChemPort |
24. Carlos, T.M. & Harlan, J.M. Leukocyte-endothelial adhesion molecules. Blood 84, 2068–2101 (1994). | PubMed  | ISI | ChemPort |
25. Plow, E.F., Haas, T.A., Zhang, L., Loftus, J. & Smith, J.W. Ligand binding to integrins. J. Biol. Chem. 275, 21785–21788 (2000). | Article | PubMed  | ISI | ChemPort |
26. Ossowski, L. & Aguirre-Ghiso, J.A. Urokinase-receptor and integrin partnership: Coordination of signaling for cell adhesion, migration and growth. Curr. Opin. Cell. Biol. 12, 613–620 (2000). | Article | PubMed  | ISI | ChemPort |
27. Preissner, K.T., Kanse, S.M. & May, A.E. Urokinase receptor: A molecular organizer in cellular communication. Curr. Opin. Cell. Biol. 12, 621–628 (2000). | Article | PubMed  | ISI | ChemPort |
28. Chapman, H.A., Wei, Y., Simon, D.I. & Waltz, D.A. Role of urokinase receptor and caveolin in regulation of integrin signaling. Thromb. Haemost. 82, 291–297 (1999). | PubMed  | ISI | ChemPort |
29. Chavakis, T. et al. Different mechanisms define the antiadhesive function of high molecular weight kininogen in integrin- and urokinase receptor-dependent interactions. Blood 96, 514–522 (2000). | PubMed  | ISI | ChemPort |
30. Chavakis, T., May, A.E., Preissner, K.T. & Kanse, S.M. Molecular mechanisms of zinc-dependent leukocyte adhesion involving the urokinase receptor and 2-integrins. Blood 93, 2976–2983 (1999). | PubMed  | ISI | ChemPort |
31. May, A.E. et al. Urokinase receptor (CD87) regulates leukocyte recruitment via 2-integrins in vivo. J. Exp. Med. 188, 1029–1037 (1998). | Article | PubMed  | ISI | ChemPort |
32. Chavakis, T. et al. Regulation of leukocyte recruitment by polypeptides derived from high molecular weight kininogen. FASEB J. 15, 2365–2376 (2001). | Article | PubMed  | ISI | ChemPort |
33. Bosse, R. & Vestweber, D. Only simultaneous blocking of the L- and P-selectin completely inhibits neutrophil migration into mouse peritoneum. Eur. J. Immunol. 24, 3019–3024 (1994). | PubMed  | ISI | ChemPort |
34. Borges, E. et al. The P-selectin glycoprotein ligand-1 is important for recruitment of neutrophils into inflamed mouse peritoneum. Blood 90, 1934–1942 (1997). | PubMed  | ISI | ChemPort |
35. Hartleib, J. et al.. Protein A is the von Willebrand factor binding protein on Staphylococcus aureus. Blood 966, 2149–2156 (2000).
36. Hussain, M. et al. Insertional inactivation of Eap in staphylococcus aureus strain Newman confers reduced staphylococcal binding to fibroblasts. Infect. Immun. in press (2002). | PubMed  |
37. Hoyer-Hansen, G., Behrendt, N., Ploug, M., Dano, K. & Preissner, K.T. The intact urokinase receptor is required for efficient vitronectin binding: Receptor cleavage prevents ligand interaction. FEBS Lett. 420, 79–85 (1997). | Article | PubMed  | ISI | ChemPort |
38. Stockmann, A., Hess, S., DeClerck, P., Timpl, R. & Preissner, K.T. Multimeric vitronectin: Identification and characterization of conformation-dependent self-association of the adhesive protein. J. Biol. Chem. 268, 22874–22882 (1993). | PubMed  | ISI | ChemPort |
39. Palma, M., Wade, D., Flock, M. & Flock, J.I. Multiple binding sites in the interaction between fibrinogen and an extracellular fibrinogen binding protein from Staphylococcus aureus. J. Biol. Chem. 273, 13177–13181 (1998). | Article | PubMed  | ISI | ChemPort |
40. Hussain, M., Becker, K., von Eiff, C., Peters, G. & Herrmann, M. Analogs of Eap protein are conserved and prevalent in clinical Staphylococcus aureus isolates. Clin. Diagn. Lab. Immunol. 8, 1271–1276 (2001). | Article | PubMed  | ISI | ChemPort |
41. Hussain, M., Hastings, J.G.M. & White, P.J. A chemically defined medium for slime production by coagulase-negative staphylococci. J. Med. Microb