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Interleukin-10 (IL-10), an anti-inflammatory cytokine, may have therapeutic potential in the fetal inflammatory response syndrome and its sequelae such as bronchopulmonary dysplasia (BPD). Our aim was to compare the effects of IL-10 versusdexamethasone (DEX) on important PMN functions of the newborn. PMNs were isolated into culture medium from cord blood after elective cesarean section deliveries. IL-10 and DEX were compared on an equimolar basis corresponding to previously measured plasma levels of DEX from infants treated for BPD. The endotoxin (LPS)-stimulated release of the pro-inflammatory cytokines, tumor necrosis factor (TNF) and IL-1, were markedly inhibited equally by IL-10 and DEX; the anti-inflammatory cytokine IL-4 was not released and IL-1 receptor antagonist (IL-1ra) was released less with DEX compared with IL-10. PMNs exposed to LPS, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), or S. aureusdid not show a significant difference between control, DEX and IL-10 for apoptosis, respiratory burst, phagocytosis or killing respectively. Chemotaxis to fMLP or IL-8 was unaffected by DEX or IL-10. The principal effects of both IL-10 and DEX, on the PMN functions studied, are related to the control of pro- and anti-inflammatory cytokine release.

An increase in polymorphonuclear leukocytes (PMNs) and proinflammatory chemokines, such as IL-8 and macrophage inflammatory protein-1 (MIP), are found in the airways during early stages of bronchopulmonary dysplasia. We determined whether IL-10 produces a dose-related inhibition of proinflammatory chemokine release from stimulated neutrophils of the newborn and whether the mechanism involves the pivotal transcription factor, nuclear factor-B. PMNs isolated from the cord blood of healthy newborns were stimulated submaximally with either lipopolysaccharide (n5) or tumor necrosis factor (n4), with and without IL-10 (0.01–1000 ng/mL). IL-8 and MIP release were measured in cell culture supernatants at 18 h. The presence or absence of nuclear factor-B activity and inhibitor-B degradation was measured at 30 min and 3 h after PMN stimulation began. During lipopolysaccharide stimulation, IL-10 significantly reduced IL-8 levels from 50 ± 16 ng/mL to 7 ± 3 ng/mL, and MIP levels from 14 ± 5 to 0.7 ± 0.1 ng/mL (mean ± SEM, p< 0.01). IL-10 produced an insignificant reduction in IL-8 and MIP levels after stimulation of PMNs with tumor necrosis factor. IL-10 did not inhibit nuclear factor-B activation and inhibitor-B degradation in PMNs stimulated with tumor necrosis factor or lipopolysaccharide for 30 min. After PMN stimulation for 3 h, inhibitor-B cytoplasmic levels were restored; however, they were unaffected by IL-10. We conclude that IL-10 is a potent inhibitor of lipopolysaccharide-stimulated release of IL-8 and MIP from neutrophils of the newborn via a mechanism not involving nuclear factor-B activity. Further work is needed to determine whether exogenous IL-10 may be useful for suppressing inflammation in bronchopulmonary dysplasia.

ABSTRACTWe have observed elevated NF-?B DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-?B activity could be correlated with a reduced level of cytoplasmic I?Bß and could be associated with the overexpression of p21CIP1/WAF1in papilloma cells. Increased NF-?B activity and cytoplasmic accumulation of p21CIP1/WAF1might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.

We have observed elevated NF-kappaB DNA-binding activity in nuclear extracts from human papillomavirus type 6- and 11-infected laryngeal papilloma tissues. The predominant DNA-binding species is the p50/p50 homodimer. The elevated NF-kappaB activity could be correlated with a reduced level of cytoplasmic IkappaBbeta and could be associated with the overexpression of p21(CIP1/WAF1) in papilloma cells. Increased NF-kappaB activity and cytoplasmic accumulation of p21(CIP1/WAF1) might counteract death-promoting effects elicited by overexpressed PTEN and reduced activation of Akt and STAT3 previously noted in these tissues.

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-B (NF-B) could explain the previous observations. NF-B is a transcription factor pivotal for expression of genes encoding inflammatory cytokines such as IL-8, but NF-B has not been previously studied in the PMN of the newborn. NF-B 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-B were identified after tumor necrosis factor- stimulation; this included the previously characterized p50/65 heterodimer and a newly identified p50/50 homodimer. Both NF-B dimers were activated by tumor necrosis factor- to significantly higher levels in the neutrophil of the newborn versusadult. An additional new finding was that pretreatment of PMN with dexamethasone (10-7M, therapeutic range) inhibited activation of both NF-B complexes in both the newborn and the adult PMN. We conclude that the increased activation of NF-B by the PMN of the newborn may play an important role in neonatal inflammatory reactions. Eventually, specific targeting of NF-B activation in the neutrophil may be an effective molecular approach for the treatment of neutrophil-mediated disorders in the newborn.

Phosphatidylinositol (4)P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2must be stringently regulated. In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe.We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombehomolog of casein kinase I. Phosphorylation by Cki1 in vitrodecreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pomberesults in a reduced synthesis of PtdIns(4,5)P2and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombeand that Cki1 is involved in regulation of PtdIns(4, 5)P2synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.

Nucleoplasmin is a phosphorylated nuclear-accumulating protein. We report herein that the kinetics of its cytoplasm r nucleus transport are affected by its degree of phosphorylation. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein; (ii) its phospho-transferase activity can be competed out by GTP; (iii) it is stimulated by polylysine; and (iv) it is inhibited by heparin. Moreover, a polyclonal antibody to the α (38 kDa) and α′ (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin’s nuclear transport. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II.