Effect of proteasome inhibitors with different chemical structures on the ubiquitin-proteasome system in vitro.

Proteasome inhibitor therapeutics (PITs) have the potential to cause peripheral neuropathy. In a mouse model of PIT-induced peripheral neuropathy, the authors demonstrated that ubiquitin-positive multifocal protein aggregates with nuclear displacement appear in dorsal root ganglion cells of animals that subsequently develop nerve injuries. This peripheral-nerve effect in nonclinical models has generally been recognized as the correlate of grade 3 neuropathy in clinical testing. In differentiated PC12 cells, the authors demonstrated perturbations correlative with the development of neuropathy in vivo, including ubiquitinated protein aggregate (UPA) formation and/or nuclear displacement associated with the degree of proteasome inhibition. They compared 7 proteasome inhibitors of 3 chemical scaffolds (peptide boronate, peptide epoxyketone, and lactacystin analog) to determine if PIT-induced peripheral neuropathy is modulated by inhibition of the proteasome (ie, a mechanism-based effect) or due to effects independent of proteasome inhibition (ie, an off target or chemical-structure-based effect). The appearance of UPAs was assayed at IC(90) +/- 5% (90% inhibition concentration +/- 5%) for 20S proteasome inhibition. Results show that each of the investigated proteasome inhibitors induced identical proteasome-inhibitor-specific ubiquitin-positive immunostaining and nuclear displacement in PC12 cells. Other agents--such as paclitaxel, cisplatin, and thalidomide, which cause neuropathy by other mechanisms--did not cause UPAs or nuclear displacement, demonstrating that the effect was specific to proteasome inhibitors. In conclusion, PIT-induced neuronal cell UPA formation and nuclear displacement are mechanism based and independent of the proteasome inhibitor scaffold. These data indicate that attempts to modulate the neuropathy associated with PIT may not benefit from changing scaffolds.

IL-8 production in human lung fibroblasts and epithelial cells activated by the Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone is transcriptionally regulated by NF-kappa B and activator protein-2.

The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides -1481 and -546 and the transcription factor NF-kappaB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-kappaB consensus DNA. The activation of NF-kappaB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.

Expression of stromal-derived factor-1 is decreased by IL-1 and TNF and in dermal wound healing.

Stromal-derived factor-1 (SDF-1) is a CXC chemokine that is believed to be constitutively expressed by stromal cells of numerous tissues. In this report, we demonstrate that dermal fibroblasts and vessels of noninflamed tissues express SDF-1. Unexpectedly, we found that expression of SDF-1 is regulated by inflammation. Expression of SDF-1 by primary cultures of human gingival fibroblasts is potently inhibited by activated macrophages via secretion of IL-1alpha and TNF-alpha. Levels of SDF-1 mRNA also decrease in acutely inflamed mouse dermal wounds. We propose that SDF-1 functions as a homeostatic regulator of tissue remodeling, whose expression stabilizes existing dermal architecture.

Maturation decreases responsiveness of human bone marrow B lineage cells to stromal-derived factor 1 (SDF-1).

We compared the chemotactic responsiveness of different subsets of human B lineage cells to stromal derived factor-1 (SDF-1). High percentages (30-40% of input) of purified bone marrow progenitors including non-B lineage progenitors, pro-B cells, and pre-B cells migrated to SDF-1alpha, demonstrating that SDF-1 is an efficacious chemoattractant of these cells. Pro-B cells responded optimally to a lower concentration of SDF-1 than other subsets, demonstrating that SDF-1 is a more potent chemoattractant of this subset. A lower percentage (10-15% of input) of mature B lymphocytes migrated to SDF-1alpha than pro-B cells, demonstrating that responsiveness of B lineage cells to SDF-1 decreases during differentiation. Inhibition by anti-CXCR4 mAb demonstrated that migration of B lineage cells to SDF-1 was completely dependent on CXC chemokine receptor-4 (CXCR4). Mature B cells expressed higher levels of CXCR4 receptors than uncommitted progenitors and pro-B cells, despite differences in responsiveness to SDF-1. CXCR4 receptors expressed by unresponsive and SDF-1-responsive B cells bound SDF-1alpha with similar affinities (K(D) = 1.7-3.3 x 10(-9) M). Therefore, elements downstream from CXCR4 appear to regulate responsiveness of B cells to SDF-1. We speculate that SDF-1 and CXCR4 direct migration of progenitor cells in microenvironments that promote B lymphopoiesis.

Regulation of IgE and cytokine production by cAMP: implications for extrinsic asthma.

Recent investigations indicate that pharmacologic agents which elevate intracellular levels of cyclic AMP (cAMP) also enhance immunoglobulin E (IgE) production. This review proposes that elevation of intracellular cAMP is a prominent mechanism which enhances IgE production. Enhancement is mediated by two mechanisms. First, cAMP-elevating agents directly target B lymphocytes, promoting recombination of the Ig heavy chain loci. Second, these agents indirectly promote IgE production by inducing a T-helper type 2 (Th2) profile of cytokine secretion. In turn, Th2-type cytokines interact with B lymphocytes and direct isotype switching to the epsilon locus. One type of cAMP-elevating agents, the beta2-adrenergic receptor agonists (beta2-agonists), are used to treat asthma. A number of detrimental phenomena have been associated with beta2-agonist use such as, rebound hyperresponsiveness and increases in asthma mortality. This review theorizes that beta2-agonists enhance IgE and Th2 cytokine production and that these mediators exacerbate extrinsic, IgE-dependent asthma.

Prostaglandin E2 receptors of the EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells.

Prostaglandin E2 (PGE2) is a potent lipid molecule with complex proinflammatory and immunoregulatory properties. PGE2 can shape the immune response by stimulating the production of IgE antibody by B lymphocytes and the synthesis of T-helper type 2 cytokines [e.g., interleukin (IL)-4, IL-10], while inhibiting production of Th1 cytokines (e.g., interferon-gamma, IL-12). It is unknown what type of receptor binds PGE2 and modulates these responses. Recent analyses in nonhematopoietic cells have identified six PGE2 receptors (EP1, EP2, EP3 alpha, EP3 beta, EP3 gamma, and EP4). This investigation examines quiescent B lymphocytes and reports that these cells express mRNA encoding EP1, EP2, EP3 beta, and EP4 receptors. The immunoregulatory functions of each receptor were investigated using small molecule agonists that preferentially bind EP receptor subtypes. Unlike agonists for EP1 and EP3, agonists that bound EP2 or EP2 and EP4 receptors strongly inhibited expression of class II major histocompatibility complex and CD23 and blocked enlargement of mouse B lymphocytes stimulated with IL-4 and/or lipopolysaccharide. PGE2 promotes differentiation and synergistically enhances IL-4 and lipopolysaccharide-driven B-cell immunoglobulin class switching to IgE. Agonists that bound EP2 or EP2 and EP4 receptors also strongly stimulated class switching to IgE. Experiments employing inhibitors of cAMP metabolism demonstrate that the mechanism by which EP2 and EP4 receptors regulate B lymphocyte activity requires elevation of cAMP. In conclusion, these data suggest that antagonists to EP2 and EP4 receptors will be important for diminishing allergic and IgE-mediated asthmatic responses.

A molecular analysis of PGE receptor (EP) expression on normal and transformed B lymphocytes: coexpression of EP1, EP2, EP3beta and EP4.

The E-series prostaglandins (PGEs) are complex lipid regulators of B lymphocyte function. They inhibit the growth of certain B lymphoma lines. We report that heterogeneity with respect to PGE-induced growth inhibition correlates with the maturation state of the B cell lines. Specifically, the pre-B cell line 70Z/3 and the immature lymphoma CH31 are extremely sensitive to PGE2. To a lesser degree, other immature lymphomas (CH33, ECH408.1 and WEHI-231) are sensitive to PGE2. More mature lymphomas (BAL-17, CH12 and CH27) and fully differentiated myelomas (J558 and MOPC-315) are insensitive to PGE2. It is unknown what subtype of PGE receptor(s) (EPs) are expressed by B lymphocytes. It is also unknown if modulation of EP receptor expression could account for the differences in the sensitivity of these B cell lines to PGE2. To investigate these issues, reverse transcriptase polymerase chain reaction, Northern blot and DNA sequencing analyses were employed to obtain a definitive EP receptor subtype profile for these B cell lines, and for normal splenic B lymphocytes. Both normal and transformed B lymphocytes express mRNA encoding EP1, EP3beta and EP4 subtypes of PGE receptors. The B lineage cells do not express EP3alpha nor EP3gamma mRNA. The B cell lines are clonal, indicating that EP1, EP3beta and EP4 mRNA are coexpressed. Surprisingly, quantitative differences in basal EP1, EP3beta and EP4 expression were not observed between B cell lines despite their differing susceptibilities to PGE-induced growth inhibition. Conversely, the polyclonal activator LPS selectively upregulates EP4 mRNA expression in the mature B cell line CH12, but not in the LPS-sensitive pre-B cell line, 70Z/3. The activator LPS does not affect EP1 nor EP3beta mRNA expression. Treatment with dbcAMP, an analog of cAMP, mimics PGE-induced growth inhibition indicating that Gs-coupled EP2 and/or EP4 receptors mediate this inhibitory signal. Indeed, EP2 agonists mimic PGE2-induced growth inhibition unlike IP, EP1 and EP3-selective agonists. These data indicate that EP2 receptors are sufficient for mediating PGE-induced growth inhibition of susceptible B lineage cells.

Reactive oxygen species and not lipoxygenase products are required for mouse B-lymphocyte activation and differentiation.

A potential role for lipoxygenase (LO) products and reactive oxygen species (ROS) in mouse B-lymphocyte activation and differentiation was investigated. Previously published investigations with the nonspecific 5-LO (EC 1.13.11.34) and 12-LO (EC 1.13.11.31) inhibitors such as nordihydroguaiaretic acid (NDGA) and 6,7-dihydroxycoumarin (Esculetin), are misleading in that they suggest lymphocyte LO activity is required for activation and differentiation of these cells. In initial support of this concept, we report that NDGA and Esculetin completely inhibited B-lymphocyte activation mediated by either membrane immunoglobulin (mIg), or the lipopolysaccharide (LPS) receptor. NDGA and Esculetin completely inhibited cell enlargement and proliferation, exhibiting half maximal inhibitory concentrations (IC50S) of approximately 1 x 10(-6) M. In contrast, the highly specific 5-LO inhibitors BAY X 1005, MK-886 and Wy 50,295 did not inhibit cell enlargement or proliferation. Moreover, 5,8,11-eicosatriynoic acid (ETI) which inhibits 5- and 12-LO, and 5, 8, 11, 14-eicosatetraynoic acid (ETYA) which inhibits all known LOs did not affect B-lymphocyte proliferation. Interestingly, NDGA and Esculetin are antioxidants, unlike BAY X 1005, MK-886, Wy 50,295, ETI and ETYA. Our hypothesis was that the antioxidant activities of NDGA and Esculetin were reponsible for inhibiting B-lymphocyte activation and proliferation and we speculated that ROS and not LO activity was required for both processes. Additional antioxidants such as butylated hydroxy toluene, o-phenanthroline, thiourea, and alpha-tocopherol (vitamin E), also inhibited B-lymphocyte proliferation induced by either the LPS or mIg receptors. These agents exhibited IC50S of 1 x 10(-8) M, 5 x 10(-10) M, 6 x 10(-3) M and 5 x 10(-5) M, respectively. When resting B-lymphocytes were treated with a source of ROS (1 x 10(-5) M H2O2), cells enlarged in a temperature-sensitive manner, which is similar to LPS-induced enlargement. Both NDGA and Esculetin completely inhibited H2O2-induced enlargement. These results further indicate that ROS are required for B-lymphocyte activation and proliferation. Similar results were obtained for B-lymphocyte differentiation. NDGA and Esculetin completely inhibited the development of plasma cells and displayed IC50S of 5 x 10(-6) M. Conversely, BAY X 1005, MK-886, Wy 50,295, ETI, and ETYA did not block the formation of plasma cells. Therefore, ROS are also crucial for differentiation into plasma cells. These experiments are the first to directly illustrate that intracellular ROS mediate B-lymphocyte activation, proliferation and differentiation and that LO products are not required for these processes.(ABSTRACT TRUNCATED AT 400 WORDS)