Michael T. Largen

Differential protein synthesis by murine peritoneal macrophages elicited by various stimuli

Protein synthetic patterns of murine peritoneal macrophages were analyzed by two‐dimensional Polyacrylamide gel electrophoresis (2D PAGE) of 35S methionine‐labeled proteins. While the protein synthetic patterns exhibited by resident, inflammatory, and activated macrophages had numerous common features that distinguished them from the other normal non‐macrophage cell types examined, unique proteins also characterized each macrophage population from the others. The accumulation by resident macrophages of proteins 23, 25, and 37 distinguished them from elicited cells, as did the formers more abundant synthesis of proteins 54 and 52. The protein synthetic patterns of inflammatory thioglycollate‐ and proteose peptone‐elicited macrophages were strikingly similar, save for the formers greater levels of accumulation of proteins 14 and 28, and the letters more pronounced expression of p23.5. Peritoneal macrophages elicited by treatment with heat‐killed Propionibacterium acnes, the live, attenuated Mycobacterium bovis strain BCG, Listeria monocytogenes, and the protozoan flagellate Trypanosoma rhodesiense, all exhibited tumoricidal activity in 16‐h or 72‐h functional assays. They shared a common protein synthetic profile that differentiated them from the synthetic patterns characteristic of the non‐tumoricidal resident and inflammatory macrophages. These tumoricidal macrophages were unique in synthesizing a protein(s) of approximate molecular weight 26,000 daltons. A time‐course study employing P. acnes‐activated peritoneal macrophages indicated that p26 accumulation decayed with tumoricidal capacity as a function of time in culture, although no direct correlation between lytic activity and p26 expression could be definitively established. Peritoneal macrophages elicited with proteose peptone were not directly tumoricidal but were rendered so upon in vitro treatment with nanogram amounts of bacterial lipopolysaccharide. The accumulation of low levels of p26 by the newly explanted proteose peptone‐elicited macrophages suggests the possibility that this protein characterizes macrophage populations primed as well as triggered for tumoricidal activity.

Identification of multiple-molecular-weight forms of thymocyte comitogenic activity from the monocyte/macrophage cell line RAW 264.7.

The cloned monocyte/macrophage cell line RAW 264.7 was investigated for interleukin 1 (IL-1) production. Of the inducers tested, bacterial lipopolysaccharide was found to be the most effective. The cyclic nucleotide analogs 8-BrcAMP and 8-BrcGMP were also tested, with only 8-BrcGMP being capable of inducing a small amount of IL-1 activity. Gel filtration studies revealed thymocyte mitogenic and comitogenic activity in three molecular-weight peaks: greater than 70,000, 30,000 to 40,000, and 12,000 to 18,000 Da. The multiple-molecular-weight forms were present when samples were prepared under serum-free conditions and also when samples were prepared and chromatographed in high ionic strength NaCl or under disulfide reducing conditions. Molecular charge heterogeneity was observed when proteins were chromatographed using column chromatofocusing (PBE 94). The intermediate-molecular-weight form eluted from the column over a pH range of 5.0 to 5.4; while the low-molecular-weight form eluted at three separate pHs: greater than or equal to 7.4 (unbound material), 5.2, and 4.8. The low-molecular-weight and intermediate-molecular-weight forms exhibited different dose-response curves when assayed under conditions used by other investigators (1 X 10(7) cells/ml; phytohemagglutinin, 1 microgram/ml), but very similar dose-response curves when assayed under conditions used by our laboratory (2 X 10(6) cells/ml; concanavalin A, 0.25 microgram/ml) in a thymocyte comitogen assay. The possible relationship of these multiple-molecular-weight species of thymocyte comitogenic activity from RAW 264.7 to other biological activities from cloned and noncloned cellular sources is discussed.