Biomedical subjects

J K Pullen

Publications and source records attributed to J K Pullen.

22 records · Page 2Linked to original sources

PubMed1989

Regulation of IL-1 and TNF-alpha expression during the differentiation of bone marrow derived macrophage.

Macrophage differentiation is accompanied by the acquisition of both Ag presentation and tumoricidal activities. In this set of experiments, the expression of IL-1 by bone marrow-derived macrophage (BMDM) was found to be highly regulated, with both the expression of IL-1 mRNA and mIL-1 appearing only at discrete stages of activation. The accumulation of IL-1 alpha (membrane) mRNA was induced by endotoxin but not IFN-gamma or CSF-1. mIL-1 was detected by D10.G4.1 T cells on BMDM only after 7 days of in vitro differentiation. Secreted IL-1 beta was detected by day 3 of culture, with enhanced production observed after activation with endotoxin. IL-1 beta mRNA was found to be constitutively expressed in BMDM as early as day 3 of culture. The expression of IL-1 beta mRNA was up-regulated by endotoxin after 30 min of exposure with maximal expression occurring after 2 to 6 h of exposure. Constitutive expression of IL-1 alpha mRNA was not detected but 1 h of endotoxin exposure resulted in the appearance of IL-1 alpha transcripts. As with IL-1 beta, TNF-alpha mRNA was also constitutively expressed during a wide time period of differentiation; however, in contrast, to IL-1 beta, TNF-alpha mRNA expression was up-regulated by both endotoxin and IFN-gamma. The expression of TNF-alpha macrophage by BMDM coincided with the acquisition of tumoricidal activity. An examination of the mRNA sequences encoding the proto-oncogenes c-myc and c-fms demonstrated the expression of c-myc only on day 3, whereas c-fms was constitutively expressed throughout the culture period. Endotoxin stimulation of BMDM resulted in a transitory increase in c-myc expression only at day 3 of culture, whereas endotoxin had no effect on c-fms expression until 7 days of culture at which time expression declined. In contrast, the expression of transferrin receptor mRNA transcripts, which were also constitutively expressed throughout the entire culture period, were not affected by stimulation with either endotoxin or IFN-gamma. These results indicate BMDM expression of the affector molecules IL-1 alpha and beta and the effector molecule TNF-alpha are regulated separately during unique "differentiation-specific" phases of development.

Animals↗

PubMed1989

Regulation of MHC gene expression during the differentiation of bone marrow-derived macrophages.

The ability of the macrophage to express class II MHC gene products appears to arise from both T-dependent and T-independent mechanisms. One mechanism by which macrophages express Ia-antigens in the absence of T-lymphocytes is postulated to be controlled by differentiation. By using a liquid bone marrow culture system, we have studied both class I and class II surface expression and mRNA accumulation during macrophage differentiation in vitro. The results demonstrated that Ia expression increased until 7 days in culture and then slowly declined. In contrast, class I expression appeared to steadily increase throughout the differentiation period. Northern blot analysis of RNA isolated from bone marrow-derived macrophages (BMDM) at various periods during culture, using E alpha, A alpha, and class I cDNA probes, correlated well with the results of Ia and H-2K surface expression. Further analysis demonstrated that the expression of Ia-antigens on BMDM was not the result of T-helper lymphocytes. This was determined by demonstrating (1) that bone marrow cultures were devoid of mature T-lymphocytes, (2) the absence of interferon (IFN)-gamma transcripts in both adherent and nonadherent populations of bone marrow cells, and (3) that the addition of anti-IFN-gamma monoclonal antibody (mAb) to the bone cultures did not alter the percentage of Ia-positive BMDM. Moreover, the addition of anti-tumor necrosis factor-alpha mAb to the bone marrow cultures had no effect on Ia expression by BMDM. Taken together, these results allow us to conclude that Ia expression by BMDM is probably not mediated via exogenous signals but rather results from an intrinsically controlled process.

Animals↗

PubMed1986

Bone marrow-derived macrophage expression of endogenous and transfected class II MHC genes during differentiation in vitro.

C57BL/6 (H-2b) mice fail to express I-E molecules on the surface of their cells and thus are unable to respond to I-E-restricted antigens such as GL phi and cytochrome c. Previous experiments in our laboratory have involved developing a system for studying differentiation of bone marrow cells into mature macrophage to gain a better understanding of class II MHC gene expression and function. In this study, we have used this system to transfect the E alpha d gene (cosmid 17.2) into C57BL/6 bone marrow cells and subsequently observed I-E expression on bone marrow-derived macrophages (BMDM) after differentiation in vitro. By using a modified calcium phosphate protocol, we found that the optimal period for transfection of the bone marrow cells was after 2 days of culture in vitro. By using the anti-I-E monoclonal antibody (Ia.7) derived from hybridoma 14-4-4, we detected the I-E molecule on the surface of transfected macrophages by a radiobinding assay and immunoprecipitation. BMDM expressed the I-E product maximally at 5 days of differentiation, and expression then declined. Furthermore, we have found that the expression of the I-E molecule on transfected macrophage was dependent upon exposure to interferon-gamma. Expression of I-E molecules was also detected by the generation of an allogeneic response. Transfected BMDM were compared with (CB6)F1 BMDM for their ability to stimulate C57BL/6 T cells and they were found to be equally effective. By using these initial findings, we hope to further optimize the conditions for insertion and expression of class II MHC genes in bone marrow cells.

Animals↗

PubMed1986

Alteration of macrophage differentiation into accessory and effector cells from exposure to dimethylnitrosamine (DMN) in vivo.

DMN exposure modulates cellular immunity through alterations in the maturation and hematopoiesis of macrophages. DMN-exposed bone marrow stem cells gave rise to increased colony-forming unit-macrophage (CFU-M) colonies while the resulting colonies produced fewer cells/colony. Bone marrow-derived macrophages phenotypically had decreased cells expressing Ia antigens or cells in the S-phase following DMN treatment. Concanavalin A-elicited peritoneal exudate cells from DMN-treated animals demonstrated an increase in the percentage of macrophages and in the number of immature, bi-nucleated cells obtained as well as a concomitant increase in the percentage of Ia antigen-expressing cells. Concanavalin A-elicited peritoneal exudate cells from DMN-exposed animals also had an increased secreted interleukin-1 activity following lipopolysaccharide stimulation without any alteration in the expression of membrane-bound interleukin-1. Thioglycolate-elicited peritoneal exudate cells from DMN-exposed animals demonstrated no changes in cellularity and only showed increases in the percentage of bi-nucleated cells. There were no alterations in the capacity of T cells obtained from DMN-treated animals to respond to either soluble (keyhole limpet hemocyanin) or allo-antigens; nor were there alterations in the capacity of these T cells to either produce or respond to interleukin-2. These findings suggest that the observed DMN-induced modulation(s) in cell-mediated immunity results from changes in macrophage hematopoiesis due to alterations in: the production of regulatory factors controlling their production and/or differentiation or their ability to respond to these factors.

Animals↗