Endothelial-neutrophil interactions during ischemia and reperfusion injury: basic mechanisms of hyperbaric oxygen.

Ischemia/reperfusion injury plays a central role in the development of tissue injury during multiple central nervous system diseases including acute stroke. Neutrophil adhesion to the endothelium indicates a major component of ischemia/reperfusion pathophysiology, and may be a target for therapeutic intervention. Hyperbaric oxygen has been documented to reduce ischemia/reperfusion injury in a number of different experimental models and in a single human randomized clinical trial. One mechanism responsible for the beneficial effect of hyperbaric oxygen in treatment of ischemia/reperfusion injury involves suppression of neutrophil-endothelial adhesion. This review intends to describe the current basic mechanisms responsible for hyperbaric oxygen-mediated inhibition of neutrophil-endothelial interactions following ischemia/reperfusion injury.

Endothelial oxidative stress activates the lectin complement pathway: role of cytokeratin 1.

Oxidative stress increases endothelial mannose-binding lectin (MBL) binding and activates the lectin complement pathway (LCP). However, the molecular mechanism of MBL binding to the endothelium after oxidative stress is unknown. Intermediate filaments have been previously reported to activate the classical complement pathway in an antibody-independent manner. We investigated whether oxidative stress increases human umbilical vein endothelial cell (HUVEC) cytokeratin 1 (CK1) expression and activates the LCP via MBL binding to CK1. Reoxygenation (3 hours, 21% O(2)) of hypoxic HUVECs (24 hours, 1% O(2)) significantly increased CK1 mRNA (in situ hybridization) and membrane protein expression [enzyme-linked immunosorbent assay (ELISA)/confocal microscopy]. Incubating human serum (HS) with N-acetyl-D-glucosamine or anti-human MBL monoclonal antibody attenuated MBL and C3 deposition on purified CK1 (ELISA). CK1 and MBL were co-immunoprecipitated from hypoxic HUVECs reoxygenated in HS. Treatment with anti-human cytokeratin Fab fragments attenuated endothelial MBL and C3 deposition after oxidative stress (ELISA/confocal microscopy). We conclude that: 1) endothelial oxidative stress increases CK1 expression, MBL binding, and C3 deposition; 2) inhibition of MBL attenuates purified CK1-induced complement activation; and 3) anti-human cytokeratin Fab fragments attenuate endothelial MBL and C3 deposition after oxidative stress. These results suggest that MBL binding to endothelial cytokeratins may mediate LCP activation after oxidative stress.

A keratin peptide inhibits mannose-binding lectin.

Complement plays a significant role in mediating endothelial injury following oxidative stress. We have previously demonstrated that the lectin complement pathway (LCP), which is initiated by deposition of the mannose-binding lectin (MBL), is largely responsible for activating complement on endothelial cells following periods of oxidative stress. Identifying functional inhibitors that block MBL binding will be useful in characterizing the role of the LCP in disease models. The human cytokeratin peptide SFGSGFGGGY has been identified as a molecular mimic of N-acetyl-D-glucosamine (GlcNAc), a known ligand of MBL. Thus, we hypothesized that this peptide would specifically bind to MBL and functionally inhibit the LCP on endothelial cells following oxidative stress. Using a BIAcore 3000 optical biosensor, competition experiments were performed to demonstrate that the peptide SFGSGFGGGY inhibits binding of purified recombinant human MBL to GlcNAc in a concentration-dependent manner. Solution affinity data generated by BIAcore indicate this peptide binds to MBL with an affinity (K(D)) of 5 x 10(-5) mol/L. Pretreatment of human serum (30%) with the GlcNAc-mimicking peptide (10-50 microg/ml) significantly attenuated MBL and C3 deposition on human endothelial cells subjected to oxidative stress in a dose-dependent manner, as demonstrated by cell surface ELISA and confocal microscopy. Additionally, this decapeptide sequence attenuated complement-dependent VCAM-1 expression following oxidative stress. These data indicate that a short peptide sequence that mimics GlcNAc can specifically bind to MBL and functionally inhibit the proinflammatory action of the LCP on oxidatively stressed endothelial cells.

Complement activation after oxidative stress: role of the lectin complement pathway.

The complement system plays an important role in mediating tissue injury after oxidative stress. The role of mannose-binding lectin (MBL) and the lectin complement pathway (LCP) in mediating complement activation after endothelial oxidative stress was investigated. iC3b deposition on hypoxic (24 hours; 1% O(2))/reoxygenated (3 hours; 21% O(2)) human endothelial cells was attenuated by N-acetyl-D-glucosamine or D-mannose, but not L-mannose, in a dose-dependent manner. Endothelial iC3b deposition after oxidative stress was also attenuated in MBL-deficient serum. Novel, functionally inhibitory, anti-human MBL monoclonal antibodies attenuated MBL-dependent C3 deposition on mannan-coated plates in a dose-dependent manner. Treatment of human serum with anti-MBL monoclonal antibodies inhibited MBL and C3 deposition after endothelial oxidative stress. Consistent with our in vitro findings, C3 and MBL immunostaining throughout the ischemic area at risk increased during rat myocardial reperfusion in vivo. These data suggest that the LCP mediates complement activation after tissue oxidative stress. Inhibition of MBL may represent a novel therapeutic strategy for ischemia/reperfusion injury and other complement-mediated disease states.

Hyperbaric oxygen downregulates ICAM-1 expression induced by hypoxia and hypoglycemia: the role of NOS.

Hyperbaric oxygen (HBO) is being studied as a therapeutic intervention for ischemia/reperfusion (I/R) injury. We have developed an in vitro endothelial cell model of I/R injury to study the impact of HBO on the expression of intercellular adhesion molecule-1 (ICAM-1) and polymorphonuclear leukocyte (PMN) adhesion. Human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell (BAEC) induction of ICAM-1 required simultaneous exposure to both hypoxia and hypoglycemia as determined by confocal laser scanning microscopy, ELISA, and Western blot. HBO treatment reduced the expression of ICAM-1 to control levels. Adhesion of PMNs to BAECs was increased following hypoxia/hypoglycemia exposure (3. 4-fold, P < 0.01) and was reduced to control levels with exposure to HBO (P = 0.67). Exposure of HUVECs and BAECs to HBO induced the synthesis of endothelial cell nitric oxide synthase (eNOS). The NOS inhibitor nitro-L-arginine methyl ester attenuated HBO-mediated inhibition of ICAM-1 expression. Our findings suggest that the beneficial effects of HBO in treating I/R injury may be mediated in part by inhibition of ICAM-1 expression through the induction of eNOS.

NF beta A, a factor required for maximal interleukin-1 beta gene expression is identical to the ets family member PU.1. Evidence for structural alteration following LPS activation.

We have previously identified and characterized the macrophage-, neutrophil- and B cell-specific nuclear factor beta A (NF beta A), which is involved in transcriptional regulation of the interleukin-1 beta (IL-1 beta) gene. NF beta A binds to a highly conserved sequence element located 6 bp upstream of the TATA motif within the IL-1 beta promoter and is required for maximal expression of the IL-1 beta gene. Here we show that NF beta A is identical to the previously identified ets gene family member PU.1. The NF beta A binding element shares 100% sequence identity with a novel PU.1 binding element recently found in the immunoglobulin J-chain promoter. Methylation interference DNA footprinting data demonstrated that NF beta A and PU.1 make identical protein/DNA contacts. In vitro synthesized PU.1 possesses a mobility and binding specificity identical to NF beta A as determined by electrophoretic mobility shift analysis (EMSA). Antisera directed against amino acids 39-55 of PU.1 recognizes NF beta A in a manner indistinguishable from PU.1 in EMSA 'supershift' studies. NF beta A and PU.1 also possess similar protein structure as determined by proteolytic clipping bandshift analysis. Furthermore, we show that PU.1 is able to transactivate an NF beta A-dependent promoter when co-transfected into HeLa cells which lack PU.1/NF beta A. EMSA studies using recombinant TATA binding protein (TBP) and PU.1 suggest that PU.1 may induce assembly of a distinct TBP-dependent complex on the IL-1 beta promoter. Finally, immunohistochemical confocal laser scanning microscopy studies suggest that LPS stimulation of RAW macrophages induces a structural change in the N-terminal transcriptional activation domain of PU.1.

The NF-beta A-binding element, not an overlapping NF-IL-6-binding element, is required for maximal IL-1 beta gene expression.

NF-beta A is a monocyte, neutrophil, and B cell-specific nuclear protein that is involved in regulation of the IL-1 beta gene. These studies further define the functional role of NF-beta A in RAW264.7 monocytic cells by using transient transfection analysis. We showed that NF-beta A was able to activate transcription from a heterologous promoter in a distance-independent and dose-dependent manner. NF-beta A also appeared to function in a positionally independent manner within the IL-1 beta cap-site proximal (CSP) promoter. NF-beta A was required for maximal IL-1 beta gene expression directed by the upstream LPS-inducible enhancer element. Deletion of the NF-beta A-binding sequence resulted in an 80% reduction in basal reporter gene activity and an 86% reduction in LPS-inducible reporter gene activity in constructs containing only the enhancer and CSP promoter. Other regulatory elements located between the enhancer and the cap site were not able to substitute functionally for the absence of NF-beta A. Recently, other investigators have reported that IL-1 beta CSP promoter function was decreased by introducing multiple mutations within both the NF-beta A-binding sequence, and a putative overlapping NF-IL-6-binding sequence. We have found that these mutations predominantly affect NF-beta A binding. Furthermore NF-beta A, and not NF-IL-6, was required for supporting basal and LPS-inducible transcription from a minimal IL-1 beta CSP promoter (positions -58 to +11). This promoter region did not appear to direct monocyte-specific IL-1 beta gene expression because reporter constructs containing the IL-1 beta CSP promoter were also active in transiently transfected HeLa cells.

An upstream protein interacts with a distinct protein that binds to the cap site of the human interleukin 1 beta gene.

Interleukin 1 beta (IL-1 beta) is a proinflammatory cytokine that exhibits a wide variety of biological activities. Genomic sequences that mediate the induction of human IL-1 beta gene transcription by lipopolysaccharide and phorbol esters are located more than 2,700 bp upstream of the transcriptional start site (cap site). These upstream elements require additional cap site-proximal (CSP) sequences which are necessary for basal transcription of the human IL-1 beta gene. In addition, these CSP sequences have been shown to mediate both cell type-specific expression of this gene, and trans-activation by some viral proteins. In this study, we report the identification of a novel nuclear protein, termed NF beta C, that binds to a DNA sequence which spans the cap site of the human IL-1 beta gene (positions -12 to +8). We have also identified a second region (positions -305 to -280) containing a putative NF-kappa B binding site. We show here that this region can bind three distinct nuclear proteins. One protein is similar or identical to NF-kappa B, a second protein (termed NF beta B) binds a distinct sequence that substantially overlaps the 5' half of the NF kappa B binding sequence, and a third protein (termed NF beta D) binds a distinct sequence that substantially overlaps the 3' half of the NF kappa B binding sequence. Unlike NF kappa B, NF1 beta B and NF beta D are present in nuclear extracts prepared from unstimulated monocytic cells. Although the NF beta D and NF beta C binding sequences share no significant similarity, each sequence can specifically compete for the binding of either protein to DNA, whereas oligonucleotides containing only the NF kappa B or NF beta B motifs do not compete for the binding of NF beta C or NF beta D. This suggests that NF beta C and NF beta D can specifically interact in vitro, possibly through a common subunit.

IL-4 reciprocally regulates IL-1 and IL-1 receptor antagonist expression in human monocytes.

Activation of human monocytes with LPS induces coordinate expression of a number of cytokine genes, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8. The T cell-derived lymphokine, IL-4, inhibits expression of these genes in monocytes, suggesting that it may be an important physiologic regulator of cytokine production. We have previously shown that IL-4 reduces steady state messenger RNA (mRNA) levels for IL-1 beta in human monocytes by decreasing both IL-1 beta transcription and the t1/2 of newly formed IL-1 beta mRNA transcripts. In the present study, we extend these findings to show that IL-4 similarly accelerates the turnover of IL-6 mRNA in LPS-stimulated monocytes. However, this inhibition of cytokine expression and dramatic increase in the decay rate of cytokine mRNA does not extend to all LPS-inducible genes because IL-4 treatment did not inhibit the expression or accelerate the turnover of mRNA for the IL-1 receptor antagonist (IL-1ra) in the same cells. Although IL-1 beta and IL-1Ra are both LPS-inducible genes, they displayed distinct temporal patterns of expression. Peak steady state mRNA levels for IL-1ra lagged significantly behind that of IL-1 beta, suggesting a possible endogenous mechanism for limiting IL-1 biologic activity. Furthermore, although IL-4 suppressed expression of both IL-1 beta and IL-6, it up-regulated synthesis of IL-1ra mRNA and protein. Thus, IL-4 inhibits production of the proinflammatory cytokine, IL-1 beta, while concomitantly enhancing synthesis of the IL-1ra in activated human monocytes.