title: Integrin regulation of polymorphonuclear leukocyte apoxis during hypoxia is primarily dependent on very late activation antigens 3 and 5.
abstract: BACKGROUND
  Apoptosis is thought to be a central mechanism that leads to resolution of the inflammatory response. The regulation of polymorphonuclear leukocyte (PMN) apoptosis during hypoxia has not been previously characterized, and we hypothesized that integrin signaling by matrix proteins (laminin) would regulate PMN apoptosis.
  
  
  METHODS
  PMNs at 1 x 10(5)/ml were adhered on plastic or laminin for 12 hours during normoxia or hypoxia. Apoptosis was determined both by cellular histologic evaluation and the TUNEL assays (Tdt). Phagocytosis in apoptotic PMNs was determined with two-color flow cytometric analyses with rhodamine-labeled heat-killed Escherichia coli (511 nm) and the Tdt reagent (563 nm). Western blot analyses were performed on nine apoptotic regulatory proteins with monoclonal antibodies directed against each protein, and tyrosine phosphorylation was assessed after integrin receptor cross-linkage.
  
  
  RESULTS
  Adherence of PMNs to laminin reduced apoptosis by cellular histologic evaluation and the Tdt method (%apoptosis = 19 +/- 1.0 versus 63 +/- 4.2 by histologic evaluation, 38 +/- 3.8 versus 60 +/- 10.5 by flow cytometry +/- adherence to laminin). Apoptosis-positive PMNs exhibited significantly greater phagocytosis than apoptosis-negative PMNs +/- laminin. Western blot analyses demonstrated increased p53 expression after 2 and 4 hours of hypoxia. Cross-linkage of very late activation antigen-3 (alpha 3/beta 1) resulted in the phosphorylation of 53 kd, 44 kd, and 39 kd proteins at 30 seconds.
  
  
  CONCLUSIONS
  (1) Chemotaxis of PMNs into the interstitium during hypoxia not only provides a means of ensuring PMN-pathogen contact but also provides a mechanism for improved survival by reducing apoptosis. (2) The reduction of apoptosis is mediated primarily by very late activation antigen-3, which leads to a subsequent increase in the intracellular expression of p53 and increased bacterial phagocytosis.
authors.affiliation: Brown University School of Medicine, Providence, R.I., USA.
authors.forename: S
authors.initials: S
authors.lastname: Leuenroth
authors.forename: E
authors.initials: E
authors.lastname: Isaacson
authors.forename: C
authors.initials: C
authors.lastname: Lee
authors.forename: H
authors.initials: H
authors.lastname: Keeping
authors.forename: H H
authors.initials: HH
authors.lastname: Simms
mesh_terms: D017209:Apoptosis; D002448:Cell Adhesion; D015687:Cell Hypoxia; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D039201:Integrin alpha3beta1; D016023:Integrins / Q000502:physiology*; D007700:Kinetics; D007797:Laminin; D009504:Neutrophils / Q000166:cytology / Q000276:immunology / Q000502:physiology*; D010587:Phagocytosis; D017469:Receptors, Fibronectin / Q000502:physiology*; D017474:Receptors, Laminin / Q000502:physiology; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
chemical_list: D039201:Integrin alpha3beta1; D016023:Integrins; D007797:Laminin; D017469:Receptors, Fibronectin; D017474:Receptors, Laminin; D016159:Tumor Suppressor Protein p53