Notch signaling mediates granulocyte-macrophage colony-stimulating factor priming-induced transendothelial migration of human eosinophils.

BACKGROUND: Priming with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances eosinophil migration and exacerbates the excessive accumulation of eosinophils within the bronchial mucosa of asthmatics. However, mechanisms that drive GM-CSF priming are incompletely understood. Notch signaling is an evolutionarily conserved pathway that regulates cellular processes, including migration, by integrating exogenous and cell-intrinsic cues. This study investigates the hypothesis that the priming-induced enhanced migration of human eosinophils requires the Notch signaling pathway. METHODS: Using pan Notch inhibitors and newly developed human antibodies that specifically neutralize Notch receptor 1 activation, we investigated a role for Notch signaling in GM-CSF-primed transmigration of human blood eosinophils in vitro and in the airway accumulation of mouse eosinophils in vivo. RESULTS: Notch receptor 1 was constitutively active in freshly isolated human blood eosinophils, and inhibition of Notch signaling or specific blockade of Notch receptor 1 activation during GM-CSF priming impaired priming-enhanced eosinophil transendothelial migration in vitro. Inclusion of Notch signaling inhibitors during priming was associated with diminished ERK phosphorylation, and ERK-MAPK activation was required for GM-CSF priming-induced transmigration. In vivo in mice, eosinophil accumulation within allergic airways was impaired following systemic treatment with Notch inhibitor, or adoptive transfer of eosinophils treated ex vivo with Notch inhibitor. CONCLUSIONS: These data identify Notch signaling as an intrinsic pathway central to GM-CSF priming-induced eosinophil tissue migration.

Eosinophil-derived cytokines in health and disease: unraveling novel mechanisms of selective secretion.

Over the past two decades, our understanding of eosinophils has evolved from that of categorically destructive effector cells to include active participation in immune modulation, tissue repair processes, and normal organ development, in both health and disease. At the core of their newly appreciated functions is the capacity of eosinophils to synthesize, store within intracellular granules, and very rapidly secrete a highly diverse repertoire of cytokines. Mechanisms governing the selective secretion of preformed cytokines from eosinophils are attractive therapeutic targets and may well be more broadly applicable to other immune cells. Here, we discuss recent advances in deciphering pathways of cytokine secretion, both from intact eosinophils and from tissue-deposited cell-free eosinophil granules, extruded from eosinophils undergoing a lytic cell death.

Immunoregulatory roles of eosinophils: a new look at a familiar cell.

Eosinophils are usually considered as end-stage degranulating effector cells of innate immunity. However, accumulating evidence has revealed additional roles for eosinophils that are immunoregulatory in nature in both the adaptive and innate arms of immunity. Specifically, eosinophils have key immunoregulatory roles as professional antigen-presenting cells and as modulators of CD4(+) T cell, dendritic cell, B cell, mast cell, neutrophil, and basophil functions. This review addresses the emerging immunoregulatory roles of eosinophils with a focus on recent data that support this new paradigm. Recognizing both the effector and immunoregulatory functions of eosinophils will enable a fuller understanding of the roles of eosinophils in allergic airways inflammation and may be pertinent to therapies that target eosinophils both for their acute and ongoing immunomodulatory functions.

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

Imaging features of intrapancreatic accessory spleen.

Although accessory spleens are commonly identified on CT, intrapancreatic accessory spleen (IPAS) is often not recognised or is mistaken for other pancreatic lesions. Currently, with improved cross-sectional techniques and spatial resolution, IPAS is more detectable. We report the imaging features and work-up for the differentiation between IPAS and other pancreatic lesions. An index case of a suspected pancreatic tail islet cell tumour, subsequently confirmed as IPAS, led to inquiries into the incidence of IPAS and the means of preventing unnecessary surgery. For 2 years, we searched for IPAS during our daily interpretations and compared these cases with those taken from our institution's database to determine the distinguishing characteristics. Three proven cases of IPAS, which mimicked pancreatic tail lesions on CT, are presented. Nine patients with suspected IPAS, based on imaging characteristics and stability, are also described. All cases of IPAS are well defined, 1-3 cm in size, follow the density and intensity of the spleen on CT and MRI, and accumulate technetium-99m ((99)Tc(m)) sulphur colloid and (99)Tc(m) heat damaged red blood cell scintigraphy (in contrast to other lesions). In conclusion, radiologists should be aware that a subtle pancreatic tail lesion could be an IPAS. A high index of suspicion will lead to correlative imaging. A combination of CT, MRI and nuclear medicine examinations can confirm the diagnosis and prevent unnecessary surgery.

Decentralization of forensic services.

Decentralization of mental health services to persons in contact with the criminal justice system is essential in the application of community mental health concepts. This is feasible considering the levels of "dangerousness" of such persons, costs of the typical isolated central facility, and implications of the Supreme Court decision Jackson v. Indiana (406 U.S. 715, 1972). In Tennessee, reform began with legal review of unit records, pretrial screening in mental health centers, revision of state law, liaison with the Tennessee Board of Pardons and Paroles, and use of consultant staff. The results are positive wherever decentralization of services has been maintained.

HeLa cell variants that differ in sensitivity to monofunctional alkylating agents, with independence of cytotoxic and mutagenic responses.

Different strains of the established human cell line HeLa differ substantially in sensitivity to ethyl methanesulfonate (EtMes). The EtMes doses effective for either cytotoxicity or mutation induction in a line of HeLa S3 cells are about 1/10th those required in the CCL2 HeLa line of the American Type Culture Collection. By plating the sensitive HeLa S3 line in the presence of highly cytotoxic doses of EtMes, we obtained a clone (designated A6) that displays about 7-fold greater resistance to EtMes toxicity. This A6 isolate is also cross resistant to other simple monofunctional alkylating agents-exhibiting about 4-fold increased resistance to methyl methanesulfonate and 10- to 15-fold increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine but is similar to the S3 parent in sensitivity to mitomycin C, UV radiation, and gamma-rays. In contrast to the results for cytotoxicity, the A6 variant and the S3 parent showed the same high susceptibility to EtMes induction of ouabain-resistant mutations. This is direct biological evidence that different alkylation lesions are normally responsible for mutagenic and cytotoxic effects. The S3 and A6 cell lines may differ in DNA repair capability specific to certain potentially lethal alkylation products. The comparative sensitivity of the A6 cells to alkylation mutagenesis may also prove useful in cell genetic studies by facilitating the generation of multiple mutants for recessive alleles and permitting exceptionally sensitive detection of specific mutagenic effects.

Mice genetically deficient in immunoglobulin E are more permissive hosts than wild-type mice to a primary, but not secondary, infection with the filarial nematode Brugia malayi.

Primary and secondary murine and human infections with Brugia malayi are characterized by substantial increases in levels of immunoglobulin E (IgE). To investigate whether this is necessary for worm clearance, IgE(-/-) mice were subjected to primary- and secondary-infection protocols. Following a primary infection, IgE(-/-) mice displayed a profound deficit in their ability to clear an intraperitoneal injection of L3 infective-stage larvae in comparison to wild-type counterparts and maintained substantial worm burdens as late as 10 weeks postinfection. Although viable adult parasites were recovered at this late time point from IgE(-/-) mice, the majority of the mice remained free of microfilariae. IgE(-/-) cohorts subjected to a secondary-infection protocol were able to clear the challenge inoculation in an accelerated manner, with kinetics similar to that observed in the wild-type animals. Analysis of the humoral response in IgE(-/-) mice following infection demonstrates a defect in IgG1 and IgG2a production, in addition to the expected lack of IgE. The IgG1 deficiency is no longer evident following a secondary infection. These data imply that deficiencies other than IgE production (i.e., IgG1 production) deficiency may be responsible for the increased permissiveness of IgE(-/-) mice as hosts following infection with B. malayi.

Dermal-resident CD14+ cells differentiate into Langerhans cells.

Epidermal Langerhans cells (LCs) show extraordinary immunostimulatory capacity and play a key role in the initiation and regulation of immune responses. Studies of LC biology are currently the focus of efforts to engineer immune responses and to better understand the immunopathology of cutaneous diseases. Here we identified and characterized a population of LC precursors that were resident in human skin. These immediate precursors expressed CD14, langerin and functional CCR6. When cultured with transforming growth factor-beta1 alone, they had the potential to differentiate into epidermal LCs; when cultured in the presence of granulocyte macrophage-colony-stimulating factor and interleukin 4 they differentiated into functionally mature dendritic cells. Identification and characterization of these LC precursors provided insight into LC biology and the mechanism(s) through which LCs repopulate the epidermis.

Direct transfection and activation of human cutaneous dendritic cells.

Gene therapy techniques can be important tools for the induction and control of immune responses. Antigen delivery is a critical challenge in vaccine design, and DNA-based immunization offers an attractive method to deliver encoded transgenic protein antigens. In the present study, we used a gene gun to transfect human skin organ cultures with a particular goal of expressing transgenic antigens in resident cutaneous dendritic cells. Our studies demonstrate that when delivered to human skin, gold particles are observed primarily in the epidermis, even when high helium delivery pressures are used. We demonstrate that Langerhans cells resident in the basal epidermis can be transfected, and that biolistic gene delivery is sufficient to stimulate the activation and migration of skin dendritic cells. RT-PCR analysis of dendritic cells, which have migrated from transfected skin, demonstrates the presence of transgenic mRNA, indicating direct transfection of cutaneous dendritic cells. Importantly, transfected epidermal Langerhans cells can efficiently present a peptide derived from the transgenic melanoma antigen MART-1 to a MART-1-specific CTL. Taken together, our results demonstrate direct transfection, activation, and antigen-specific stimulatory function of in situ transduced human Langerhans cells.