Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment.

Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-alpha (an E-selectin inducer) and cycloheximide also eliminated E-selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P-selectin expression to support adequate leukocyte recruitment.

A role for platelets and endothelial selectins in tumor necrosis factor-alpha-induced leukocyte recruitment in the brain microvasculature.

The mechanisms mediating leukocyte recruitment into the cerebral nervous system during inflammation are still poorly understood. The objective of this study was to investigate the leukocyte recruitment in the brain microcirculation by intravital microscopy. Superfusion of the brain with artificial cerebrospinal fluid did not induce leukocyte rolling or adhesion. However, intraperitoneal tumor necrosis factor-alpha (TNF-alpha) caused marked leukocyte rolling and adhesion in the brain microcirculation. Histology revealed that the recruitment was primarily of neutrophils. Both E- and P-selectin were required for TNF-alpha-induced leukocyte recruitment, as rolling was reduced after treatment with either anti-E- or anti-P-selectin antibody and eliminated in E- or P-selectin-deficient mice. A significant increase in brain P- and E-selectin expression was seen after TNF-alpha treatment, but both were an order of magnitude less than in any other tissue. We observed significant platelet paving of TNF-alpha-stimulated endothelium and found that anti-platelet antibody reduced leukocyte rolling and adhesion, as did acetylsalicylic acid (aspirin). However, depletion of platelets did not reduce cerebral P-selectin expression. Moreover, chimeric mice lacking P-selectin on endothelium but not platelets had significantly decreased P-selectin expression and reduced leukocyte recruitment in the brain. This suggests a role for endothelial P-selectin in cerebral leukocyte recruitment. In conclusion, TNF-alpha-induced neutrophil recruitment into the brain requires both endothelial E-selectin and P-selectin as well as platelets, but platelet P-selectin was not a major contributor to this process.

Assessment of the mechanism of juxtacrine activation and adhesion of leukocytes in liver microcirculation.

Leukotriene C4 (LTC4), histamine, and other mediators can induce expression of P-selectin and platelet-activating factor (PAF) on venular endothelium to recruit leukocytes in vivo and in vitro via a juxtacrine mechanism of adhesion. The objective of this study was to assess the effect of histamine and LTC4 on the leukocyte recruitment in the liver and to study the components and molecular mechanisms involved in this process. We visualized the hepatic microvasculature using intravital microscopy and we determined that LTC4 (20 nM) but not histamine (0.1, 0.3, or 1 mM) induced leukocyte recruitment in the liver microcirculation. Histamine could induce leukocyte recruitment but only in the presence of an antihistaminase. The LTC4-induced leukocyte recruitment occurred primarily in sinusoids (not venules) and was not inhibitable by three different anti-P-selectin antibodies (5H1, RMP-1, and RB40). Leukocyte recruitment in P-selectin-deficient mice, intercellular adhesion molecule 1 (ICAM-1)-deficient mice, and mice treated with a PAF antagonist was of the same magnitude as in wild-type animals in response to LTC4. Although PAF alone could induce adhesion in both sinusoids and postsinusoidal venules, this chemotactic agent was not involved in LTC4-induced adhesion in the liver. Finally, an overlapping role for P-selectin and ICAM-1 was ruled out as LTC4 induced leukocyte recruitment in P-selectin and ICAM-1 double-deficient mice. These data demonstrate that LTC4 does not activate the known early mechanisms of leukocyte recruitment, including P-selectin, PAF, or ICAM-1 in the hepatic microvasculature.

Immunoglobulin E-mediated anaphylaxis activates nuclear factor kappaB in rat small intestine.

OBJECTIVE AND DESIGN: Since IgE-dependent reactions induce the inducible isoform of NO synthase, we postulated the involvement of the transcription factor NF-kappaB. MATERIALS: 72 Wistar rats were divided into 6 groups and used to study the hemorrhagic necrosis of the small intestine elicited by anaphylaxis. TREATMENT: Passive anaphylaxis was produced by i.p. sensitization with IgE anti-dinitrophenyl monoclonal antibody and i.v. challenge with the cognate antigen. METHODS: Competitive PCR was used to assay the expression of p50 subunit of NF-kappaB. kappaB-binding activity was assayed by electrophoretic mobility shift assay. RESULTS: The PCR assay showed a time-dependent increase of mRNA coding for the p50 subunit of NF-kappaB, which was maximal 1 h after challenge (40+/-3, versus 230+/-32 fM, mean +/- SEM) and decreased to prechallenge level at 4h. kappaB-binding activity was also increased. CONCLUSIONS: IgE-mediated reactions trigger a pathway for nuclear signaling that seems to be related to the intermediate/late response of the allergic reaction.