 |
|
Search PubMed for publications by Dr William Bowers |
|
|
Bowers WE Christian de Duve and the discovery of lysosomes
and peroxisomes. Trends Cell Biol 1998 Aug;8(8):330-3. Short article |
|
|
Ye Z, Gee AP, Bowers WE, Lamb LS, Turner MW, Henslee-Downey PJ In
vitro expansion and characterization of dendritic cells derived from human bone marrow
CD34+ cells. Bone Marrow Transplant 1996 Nov;18(5):997-1008 Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses in vivo and in vitro, and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg GVHD and graft-versus-leukemia. Here, we describe a two-stage cell culture system for expansion of functionally active human DC from CD34+ marrow precursors. Optimal outgrowth was achieved by initially culturing CD34+ cells for 5 days in medium containing GM-CSF, MGF and TNF-alpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultures contained approximately 40% DC as determined by immunophenotype and morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a+ cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L. Reversing the sequence of growth factors during culture and subculture decreased the yield and purity of DC. Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34+ marrow precursors. |
|
|
Summary Dendritic cells (DC) found in the periphery of the cornea have been known to be important regulators of immune responses in the anterior segment of the eye. In this study, DC have been immunomagnetically isolated from rat corneal epithelium and assayed for their ability to stimulate lymphocytes. DC enriched immunomagnetically from rat corneal epithelium comprised about 1.1± 0.6% (n=14) of the total corneal cells and were able to stimulate allogeneic or periodate-treated lymphocytes, and present myelin basic protein and ovalbumin to antigen-primed T cells. Other cells had negligible lymphostimulatory activity. The functional activities of corneal DC did not increase significantly after culture for 1 or 3 days. Lymphostimulatory activity was also examined after trauma to the cornea, which caused DC to migrate into the central cornea. Confocal microscopy of corneal epithelium immunofluorescently stained with a panel of anti-DC mAb confirmed that traumatized eyes contained DC in the central cornea, whereas DC in the normal contralateral eye were located only in the peripheral corneal epithelium. DC isolated immunomagnetically from traumatized corneas had significantly greater activity in stimulating allogeneic or periodate-treated lymphocytes than did those from normal cornea. DC from the periphery and central cornea of traumatized eyes showed the same increased activity. In an attempt to delineate the factors that influence DC activity, immunomagnetically purified cells were cultured with a variety of cytokines or metabolic inhibitors for 72 hours. IL-1 and GM-CSF increased DC accessory activity by 3- and 13-fold respectively. These functional increases were inhibited with cycloheximide. These findings suggest that cytokines such as IL-1 and GM-CSF may in involved in the functional activation of DC that occurs when DC migrate to the central cornea after trauma. |
|
|
Choudhury, A., Pakalnis, V.A. and Bowers, W.E. Characterization and Functional Activity of Dendritic Cells from Rat Choroid. Exp. Eye Res., 59:297-304, 1994. Summary Cell preparations from the poster-ior eye cup of the eye cultured for two days exhibited accessory activity for T cell responses to a mitogenic treatment and stimulatory activity in a mixed leukocyte reaction (MLR), two functions characteristic of dendritic cells. These activities both partitioned with cells having a low buoyant density, another characteristic of dendritic cells. Immunomagnetic separations with mono-clonal antibodies against lymphoid dendritic cell surface antigens revealed that the accessory activity of the low-density cells was entirely associated with a small population of cells positively selected by these antibodies. Immunofluorescent staining with these same antibodies also revealed a small subpopulation of low-density cells having the morphology of dendritic cells. On cryostat sections of eye tissue the positively-stained cells were localized in the choroid and were not observed within the sclera or the retina. Based on these results we conclude that there are functional dendritic cells in the choroid, and we speculate that they may have a significant role in the inflammatory process during posterior uveitis.
|
|
|
Bowers,W.E., Yu,S., and Khandkar, S. Differentiation of Dendritic Cells in Cultures of Rat and Mouse Bone Marrow Cells. Adv. Exp. Med. Biol. 329:251-255, 1993 Summary Similar results have been obtained for the differentiation of
dendritic cells in cultures of bone marrow cells from both rat and mouse. Despite a bone
marrow origin, dendritic cells were not observed in fresh preparations of bone marrow
cells or detected by a sensitive functional assay for accessory activity. After four days
of culture in a serum-free medium, identifiable dendritic cells with readily detectable
accessory activity appeared. Addition of a Con A supernatant to cultures of bone marrow
cells had two significant effects: 1) the number of dendritic cells produced after four
days increased 5-20-fold, and, 2) the accessory activity per dendritic cell, measured with
periodate-treated T lymphocytes as responders, increased 7-10-fold. The dendritic cells
produced in culture expressed markers of dendritic cells isolated from tissues and also
had a low buoyant density. Dendritic cells were produced after division of precursors.
However, precursors had a different phenotype; in mouse they were Ia-, J11D-,
and 33D1-, and in rat they were Ia- and negative for three
monoclonal antibodies we developed that stain both lymphoid dendritic cells and dendritic
cells produced in cultures of bone marrow cells. Recombinant cytokines were tested for
their ability to augment the number and accessory activity of dendritic cells.
GM-CSF was
the only cytokine active in our system. G-CSF, M-CSF, IL-1, and IL-3 individually produced
changes only slightly above control, and combinations of cytokines has no effect unless
GM-CSF was present. We conclude that bone marrow dendritic cell precursors, which lack the
phenotype and function of dendritic cells, respond directly or indirectly to GM-CSF to
divide and differentiate into dendritic cells having phenotypic and functional attributes
similar to those isolated from tissues. |
|
|
Rochester, C.L., Goodell, E.M., Stoltenborg, J.K., and Bowers, W.E. Dendritic Cells From Rat Lung are Potent Accessory Cells. Am. Rev. Respir. Dis. 138:121-128, 1988. Summary Accessory cells are required for the induction of lymphocyte proliferation in response to mitogens or antigens. Rat pulmonary cells were tested for the presence of accessory activity in lymphocyte proliferation induced by sodium periodate. Buffer-perfused lungs were excised, minced and enzymatically digested. The resulting pulmonary cells were separated into high density (32 to 57%) and low density (9 to 32%) fractions in a discontinuous density gradient of bovine plasma albumin. Both macrophages and dendritic cells were observed in the low density cells by light microscopy. High density, low density and adherent and non adherent low density cells, as well as a purified preparation of pulmonary dendritic cells were tested for accessory activity in the presence of periodate-treated, lymphnode-derived lymphocytes as responders. Most of the accessory activity was found in the low density cells. Increasing numbers of low density cells stimulated proliferation of responder lymphocytes in a linear, dose-dependent manner; higher numbers had a suppressive effect. Nonadherent low density cells containing dendritic cells also produced a dose-dependent increase in periodate-induced lymphocyte proliferation, whereas adherent low density cells, morphologically identified as macrophages, were suppressive. Removal of phagocytic macrophages from nonadherent low density cells resulted in an eightfold increase in both the percent of dendritic cells present and the amount of accessory activity in the low density cells. We conclude that pulmonary dendritic cells are potent accessory cells for perio-date-induced lymphocyte proliferation. |
|
| Return to Dr Bowers' Home Page | |
 |
|
