IntroductionSepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro.MethodsThe concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm2).ResultsEight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either β2 (CD18), αL/β2 (CD11α/CD18; LFA-1) or αM/β2 (CD11β/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion.ConclusionsHuman SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a β2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

The mechanisms of severe sepsis induced activation/dysfunction of the cerebrovascular endothelium are poorly understood. Our findings indicate that severe sepsis in humans results in up‐regulation of 8 (p<0.01; out of 41 measured) inflammation‐relevant analytes in the blood plasma. We employed Severe sepsis Cytomix (SS‐CM) consisting of 8 cytokines/chemokines (at the levels detected in Severe sepsis‐plasma) to assess activation/dysfunction of human‐derived cerebrovascular endothelial cells (hCMEC/D3; provided by Dr. P.O. Couraud, INSERM) and neutrophilic leukocytes (PMN). Various inflammation‐relevant endpoints were assessed. The obtained results indicate that stimulation of hCMEC/D3 with SS‐CM failed to induce ROS production, activation of NF‐κB, and increase in hCMEC/D3 permeability; however ICAM‐1 gene expression was up‐regulated in hCMEC/D3. The latter was accompanied by increased PMN adhesion to hCMEC/D3 under conditions of “flow” (0.7 dyn/cm2 shear stress). The most potent increase in PMN adhesion, however, occurred when both PMN and hCMEC/D3 were stimulated with SS‐CM. PMN adhesions was prevented by interfering (neutralizing MAb) with β‐2‐integrin function. Taken together our findings indicate that increased adhesive interaction between circulating PMN and cerebrovascular endothelium may contribute to BBB dysfunction in severe sepsis via β‐2‐integrin. HSFO NA‐6914, PSI 11–01)