RESUMEN
The relative contributions of CD11a/CD18 and CD11b/CD18 to the dynamics and strength of neutrophil adhesion to intercellular adhesion molecule (ICAM)-1-transfected cells were examined over the time course of chemotactic stimulation. Suspensions of neutrophils and transfectants were sheared in a cone-plate viscometer, and formation of heterotypic aggregates was measured by 2-color flow cytometry. The 2-body collision theory was used to compute adhesion efficiency, defined as the proportion of collisions between neutrophils and target cells that resulted in capture. ICAM-1 surface density and shear rate both regulated adhesion efficiency. Target cells expressing approximately 1000 ICAM-1 sites/microm(2) (I(low)) were captured with an efficiency of 0.15 at 100 s(-1), which decreased to zero at 300 s(-1). At 8-fold higher ICAM-1 expression (I(high)) corresponding to levels measured on interleukin-1-stimulated endothelium, efficiency was 0.3 at 100 s(-1) and remained above background to 900 s(-1). Shear alone was sufficient for CD11a/CD18-mediated adhesion to ICAM-1, and stimulation with formyl-methionyl-leucyl-phenylalanine boosted capture efficiency through CD11a/CD18 by 4-fold. In comparison, CD11b/CD18 supported one third of this efficiency, but was necessary for aggregate stability over several minutes of shear and at shear stresses exceeding 5 dyne/cm(2). Hydrodynamics influenced capture efficiency predominantly through the collisional contact duration, predicted to be approximately 9 milliseconds for successful capture of I(low) and 4 milliseconds for I(high). The implication is that an increase in ICAM-1 from resting levels to those on inflamed endothelium effectively increases the permissible shear in which capture through beta(2)-integrins may occur. Neutrophil adhesion to ICAM-1 appears to be a cooperative and sequential process of CD11a-dependent capture followed by CD11b-mediated stabilization.
Asunto(s)
Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Antígeno de Macrófago-1/metabolismo , Neutrófilos/metabolismo , Adhesión Celular , Fenómenos Químicos , Química Física , Factores Quimiotácticos/farmacología , Citometría de Flujo , Hemorreología , Humanos , Cinética , Modelos Químicos , N-Formilmetionina Leucil-Fenilalanina/farmacología , Unión Proteica , Estrés Mecánico , Vasculitis/metabolismoRESUMEN
While p53 activity is critical for a DNA damage-induced G(1) checkpoint, its role in the G(2) checkpoint has not been compelling because cells lacking p53 retain the ability to arrest in G(2) following DNA damage. Comparison between normal human foreskin fibroblasts (HFFs) and HFFs in which p53 was eliminated by transduction with human papillomavirus type 16 E6 showed that treatment with adriamycin initiated arrest in G(2) with active cyclin B/CDC2 kinase, regardless of p53 status. Both E6-transduced HFFs and control (LXSN)-transduced cells maintained a prolonged arrest in G(2); however cells with functional p53 extinguished cyclin B-associated kinase activity. Down regulation was mediated by p53-dependent transcriptional repression of the CDC2 and cyclin B promoters. In contrast, cells lacking p53 showed a prolonged G(2) arrest despite high levels of cyclin B/CDC2 kinase activity, at least some of which translocated into the nucleus. Furthermore, the G(2) checkpoint became attenuated as p53-deficient cells aged in culture. Thus, at late passage, E6-transduced HFFs entered mitosis following DNA damage, whereas the age-matched parental HFFs sustained a G(2) arrest. These results indicate that normal cells have p53-independent pathways to maintain DNA damage-induced G(2) arrest, which may be augmented by p53-dependent functions, and that cells lacking p53 are at greater risk of losing the pathway that protects against aneuploidy.