Dennis Davidson

Kringle 5 of Plasminogen is a Novel Inhibitor of Endothelial Cell Growth

Angiostatin is a potent angiogenesis inhibitor which has been identified as an internal fragment of plasminogen that includes its first four kringle modules. We have recently demonstrated that the anti-endothelial cell proliferative activity of angiostatin is also displayed by the first three kringle structures of plasminogen and marginally so by kringle 4 (Cao, Y., Ji, R.-W., Davidson, D., Schaller, J., Marti, D., Sohndel, S., McCance, S. G., O’Reilly, M. S., Llinás, M., and Folkman, J. (1996) J. Biol. Chem. 271, 29461–29467). We now report that the kringle 5 fragment of human plasminogen is a specific inhibitor for endothelial cell proliferation. Kringle 5 obtained as a proteolytic fragment of human plasminogen displays potent inhibitory effect on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately 50 nm. Thus, kringle 5 would appear to be more potent than angiostatin on inhibition of basic fibroblast growth factor-stimulated capillary endothelial cell proliferation. Appropriately folded recombinant mouse kringle 5 protein, expressed inEscherichia coli, exhibits a comparable inhibitory effect as the proteolytic kringle 5 fragment. Thus, kringle 5 domain of human plasminogen is a novel endothelial inhibitor that is sufficiently potent to block the growth factor-stimulated endothelial cell growth.

NF-κB Activation in Tumor Necrosis Factor α-stimulated Neutrophils Is Mediated by Protein Kinase Cδ CORRELATION TO NUCLEAR IκBα

The transcription factor NF-κB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-κB activation in human neutrophils in response to stimulation with tumor necrosis factor-α (TNFα) are undefined. Since recent studies implicated activation of NF-κB as well as protein kinase C-δ (PKCδ) in neutrophil apoptosis, we investigated involvement of PKCδ in the activation of NF-κB in TNFα-stimulated neutrophils. Specific inhibition of PKCδ by rottlerin prevented IκBα degradation and NF-κB activation in TNFα-stimulated neutrophils. This regulation of NF-κB activation by PKCδ was specific only for TNFα signaling, since lipopolysaccharide- or interleukin-1β-induced NF-κB activation and IκBα degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IκBα in the nucleus, as well as in the cytoplasm, correlates with the NF-κB DNA binding. These results suggest that in human neutrophils, the presence of IκBα in the nucleus may function as a safeguard against initiation of NF-κB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-κB regulation.

NF-kappaB Activation in TNFalpha-Stimulated Neutrophils is Mediated by Protein Kinase C-delta: Correlation to Nuclear I-kappaB-alpha

The transcription factor NF-κB is critical for the expression of multiple genes involved in inflammatory responses and apoptosis. However, the signal transduction pathways regulating NF-κB activation in human neutrophils in response to stimulation with tumor necrosis factor-α (TNFα) are undefined. Since recent studies implicated activation of NF-κB as well as protein kinase C-δ (PKCδ) in neutrophil apoptosis, we investigated involvement of PKCδ in the activation of NF-κB in TNFα-stimulated neutrophils. Specific inhibition of PKCδ by rottlerin prevented IκBα degradation and NF-κB activation in TNFα-stimulated neutrophils. This regulation of NF-κB activation by PKCδ was specific only for TNFα signaling, since lipopolysaccharide- or interleukin-1β-induced NF-κB activation and IκBα degradation were not inhibited by rottlerin. In addition, we show that in human neutrophils, but not monocytes, IκBα localizes in significant amounts in the nucleus of unstimulated cells, and the amount of IκBα in the nucleus, as well as in the cytoplasm, correlates with the NF-κB DNA binding. These results suggest that in human neutrophils, the presence of IκBα in the nucleus may function as a safeguard against initiation of NF-κB dependent transcription of pro-inflammatory and anti-apoptotic genes, and represents a distinct and novel mechanism of NF-κB regulation.

NF-κB Regulation in Human Neutrophils by Nuclear IκBα: Correlation to Apoptosis

Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-κB plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-κB inhibitor, IκBα, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of IκBα in human neutrophils and to determine whether increased accumulation of IκBα in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of IκBα that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of IκBα that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of IκBα in stimulated neutrophils by blocking the IκBα nuclear export, and this is associated with inhibition of NF-κB activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-κB regulation in human neutrophils by nuclear IκBα. Our results demonstrate that the NF-κB activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of IκBα in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.

Okadaic acid induces sustained activation of NFκB and degradation of the nuclear IκBα in human neutrophils

Abstract Human neutrophils differ from other cells by containing high amount of IκBα in the nucleus, and this increased nuclear IκBα accumulation is associated with the inhibition of NFκB activity and increased apoptosis. However, the mechanisms regulating NFκB activation and IκBα degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFκB activity and IκBα degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFκB and degradation of the nuclear IκBα, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-δ (PKCδ) and IκB kinase (IKK) inhibit the OA-induced activation of NFκB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFκB is regulated by a continuous phosphorylation and degradation of the nuclear IκBα.

Mechanism for Dexamethasone Inhibition of Neutrophil Migration upon Exposure to Lipopolysaccharide in Vitro : Role of Neutrophil Interleukin-8 Release

The mechanism by which dexamethasone (DEX) inhibits neutrophil (PMN) recruitment to a site of inflammation, such as the newborn lung with bronchopulmonary dysplasia, is not completely understood. The aim of our study was to determine whether DEX inhibits neutrophil-induced neutrophil recruitment by inhibition of interleukin- (IL) 8 release from PMNs, and if there are developmental differences. PMNs isolated from cord blood (CB) and adults (A) were studied. We first measured the effect of DEX (10−10 to 10−4 M) on PMN migration to an exogenous IL-8 standard (10−8 M) using PMNs of CB (n= 3) and A (n= 3), over 1 h in a chemotaxis chamber. Second, we determined the effect of DEX (0 and 10−10 to 10−6 M) on IL-8 release (immunoasay) from PMNs of CB (n= 7) or A (n= 7) after incubation with lipopolysaccharide (LPS, 1 ng/mL) for 6 and 18 h. Third, the chemoattractant activity of culture media from the second experiment was studied with and without IL-8 antibody. DEX at concentrations of 10−10 to 10−4 M had no direct effect on PMN migration in vitro to an exogenous IL-8 standard. After LPS exposure, IL-8 release was greatly increased for PMNs from CB compared with A. DEX (10−10 to 10−4 M) resulted in a dose-dependent inhibition of IL-8 release from PMNs exposed to LPS for 6 and 18 h incubation. Increased PMN migration activity was only found with media of PMNs of CB with no DEX. At 18 h, media-induced migration activity was decreased if DEX (10−7 M), IL-8 antibody, or DEX (10−7 M) with IL-8 antibody were present during the incubation with LPS: there was an 88, 86, and 101% reduction in migration activity, respectively. We conclude that DEX inhibits PMN-induced PMN migration, predominantly via inhibition of IL-8 release for PMNs of the newborn. We suggest that a 10-fold lowering of the standard DEX dose may effectively reduce lung inflammation in bronchopulmonary dysplasia.

Endothelium-Derived Relaxing Factor: Presence in Pulmonary and Systemic Arteries of the Newborn Guinea Pig

ABSTRACT: Endothelium-derived relaxing factor (EDRF), believed to be nitric oxide or a compound that releases nitric oxide, is a potent vasodilator produced by some arteries in response to acetylcholine (ACh) and bradykinin (BK). ACh and BK are potent dilators of perinatal pulmonary and systemic arteries. The objectives of this study were to determine if EDRF is present in newborn vessels and if EDRF mediates the vasodilator actions of ACh and BK. Arterial rings from newborn guinea pigs, 1 to 3 d old, were obtained from a branch of the main pulmonary artery and the descending aorta for isometric force bioassays. At their optimal resting tension, the rings were preconstricted with phenylephrine 10-5 M in Krebs- Henseleit solution before adding incremental doses of ACh or BK. If the endothelium was intact, ACh (10s M) relaxed pulmonary arteries and aortas (64 ± 7%, 72 ± 9% relaxation, respectively, mean ± SE). ACh-induced relaxation (ACh 10-5 M) in the pulmonary artery and aorta, respectively, was significantly (p < 0.05) attenuated by 1) endothelial removal (11 ± 9%, 28 ± 10%) by rubbing the ring lumen; 2) methylene blue, 10-6 M, (6 ± 8%, 7 ± 3%) that inhibits EDRF-associated cGMP production in smooth muscle; and 3) methemoglobin, 10-5 M, (13 ± 9%, 17 ± 7%) that binds EDRF. The results for BK were similar to ACh for the pulmonary artery but BK did not relax the aorta. Indomethacin diminished relaxation of the pulmonary artery and aorta to the submaximal dose (10-5M) of ACh but indomethacin did not effect the relaxation to ACh 10-4 M or BK. We conclude that EDRF is produced in the guinea pig pulmonary artery and descending aorta at birth and that EDRF is a mediator of the vasodilator actions of ACh and BK. Vasodilation by ACh may also involve activation of the cyclooxygenase pathway.

Activation of Nuclear Factor-κB and Its Suppression by Dexamethasone in Polymorphonuclear Leukocytes: Newborn Versus Adult

Polymorphonuclear leukocytes (PMN) of the newborn, to a greater extent than those of the adult, have the ability to amplify PMN recruitment to an inflammatory site by their own release of IL-8, and this process is inhibited by dexamethasone. The aim of the present study was to determine whether the regulation of nuclear factor-κB (NF-κB) could explain the previous observations. NF-κB is a transcription factor pivotal for expression of genes encoding inflammatory cytokines such as IL-8, but NF-κB has not been previously studied in the PMN of the newborn. NF-κB activation was measured by an electrophoretic mobility shift assay in nuclear extracts prepared from PMN isolated from adults and cord blood from newborns. Two distinct molecular forms of NF-κB were identified after tumor necrosis factor-α stimulation; this included the previously characterized p50/65 heterodimer and a newly identified p50/50 homodimer. Both NF-κB dimers were activated by tumor necrosis factor-α to significantly higher levels in the neutrophil of the newborn versus adult. An additional new finding was that pretreatment of PMN with dexamethasone (10-7 M, therapeutic range) inhibited activation of both NF-κB complexes in both the newborn and the adult PMN. We conclude that the increased activation of NF-κB by the PMN of the newborn may play an important role in neonatal inflammatory reactions. Eventually, specific targeting of NF-κB activation in the neutrophil may be an effective molecular approach for the treatment of neutrophil-mediated disorders in the newborn.

Gene-Specific Repression of Proinflammatory Cytokines in Stimulated Human Macrophages by Nuclear IκBα

We have previously shown that increased nuclear accumulation of IκBα inhibits NF-κB activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of IκBα can be used as a therapeutic target for the regulation of NF-κB–dependent cytokine synthesis. Treatment of LPS-stimulated human U937 macrophages with an inhibitor of chromosome region maintenance 1-dependent nuclear export, leptomycin B, resulted in the increased nuclear accumulation of IκBα and inhibition of NF-κB DNA binding activity, caused by the nuclear IκBα-p65 NF-κB interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-α, the β form of pro-IL-1 (IL-1β), and IL-6 were inhibited by the leptomycin B-induced nuclear IκBα, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-κB to NF-κB–regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-α, IL-1β, and IL-6 promoters was inhibited by the nuclear IκBα, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using IκBα and S536 phosphospecific p65 NF-κB Abs demonstrated that although the newly synthesized IκBα induced by postinduction repression is recruited to TNF-α, IL-1β, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-α, IL-1β, or IL-6 promoters. Together, these data indicate that the inhibition of NF-κB–dependent transcription by nuclear IκBα in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-κB.

Comparative differences and combined effects of interleukin-8, leukotriene B4, and platelet-activating factor on neutrophil chemotaxis of the newborn

ABSTRACT: IL-8, leukotriene B4 (LTB4), and platelet-activating factor (PAF) are potent neutrophil chemoattractants that have been identified in inflammatory conditions of the newborn such as chronic lung disease of extreme prematurity. The aims of this study were to determine the relative potency and combined effects of these mediators on chemotaxis of polymorphonuclear leukocytes (PMN) from the newborn and to compare the effect of combining all three mediators on chemotaxis of PMN from newborns and adults. Neutrophils were isolated from cord blood (n = 17) or healthy adults (n = 4) and incubated in a 3-tier, 48-well chemotaxis chamber. For PMN from newborns, using chemoattractant concentrations ranging from 0.01 to 100 nM, we found that there were significant differences in potency: IL-8 > LTB4 > PAF. Migration to each of these mediators was almost completely due to chemotaxis as opposed to chemokinesis. At submaximal chemotaxis, using equally effective doses of IL-8 (0.2 nM), LTB4 (1.0 nM), and PAF (10 nM), the combination of all three mediators increased chemotaxis 2.4-fold above the average individual responses. Further studies indicated this increase in chemotaxis was due to the combination of IL-8 and PAF or IL-8 and LTB4; but there was no increase in chemotaxis when PAF and LTB4 were combined. The combination of all three submaximal doses of chemoattractants resulted in PMN chemotaxis that was still 36% of the adult response. We conclude that for neutrophils from the newborn: 1) IL-8 is a potent stimulus that has additive effects on chemotaxis in combination with either LTB4 or PAF, 2) the combination of LTB4 and PAF did not have an additive effect on chemotaxis, and 3) in spite of enhanced chemotaxis by more than one stimuli, the response is still significantly lower than for neutrophils from adults.