These studies examine the interactions of plasma, leukocytes and/or various mediators (e.g., cytokines, peptides) with cerebral endothelial cells (EC) and extracellular matrix (ECM). The investigations address in the cellular state of tolerance to brain injury. Investigation of the effect of hypoxia on neutrophil (N) interaction with ECM has shown that N adhesion is controlled by at least two different mechanisms: a) hypoxia-sensitive, CD11b-dependent and NO-independent and b) hypoxia- insensitive, CD11b-independent and NO-dependent. It was hypothesized that decreased adhesion and spreading of hypoxic neutrophils could promote N migration on ECM. N migration was recorded with a videomicroscope and then analyzed using the NIH Image computer program. Mean migration distances were 48.1 plus/minus 19.7 and 85.7 plus/minus 45.6 T for N adherent to fibronectin and laminin respectively. Hypoxia significantly potentiated neutrophil migration on both surfaces (66.5 plus/minus 26.8 and 126.2 plus/minus 50.2 T plus/minus SD; P = 0.03 and was comparable to that of 10 nM FMLP. Potentiation of N migration by hypoxia was completely abolished by a culture medium conditioned by rat cortical astrocytes activated with TNF-alpha for 48 hours but not in the absence of TNF. It is suggested that the neutrophil migration inhibitor released by TNF-alpha treated astrocytes might be one of the mediators of TNF-alpha-induced tolerance to stroke. It was previously shown that both intercellular adhesion molecule-1 (ICAM-1) expression by rat cerebral microvascular endothelial cells (EC) and monocyte (MO) adhesion to EC were dose-dependently increased by culturing EC with plasma from hibernating (HP) and to a lesser extent, non-hibernating (NHP) thirteen- lined ground squirrels. Separation of plasma by HPLC resulted in identification of fractions (MW greater than 70 kD) with potent effects on adhesive interactions between MO and EC. The preliminary data indicate that 78% of adhesion-promoting activity is located in this fraction. Additional experiments examined the effects of MO treatment with HP and NHP which inhibited MO adhesion 58% and 40%, respectively.
