Immunophenotypical changes of neoplastic cells and tumor-associated macrophages in a rat dendritic cell sarcoma-derived transplantable tumor line (KB-D8)

Abstract

Basically, dendritic cell-derived sarcomas are characterized by expression of major histocompatibility complex class-II molecules, but the biological properties of the tumor cells remain to be elucidated. Recently, we established a novel transplantable cell line (KB-D8) from a dendritic cell sarcoma found in an F344 rat. In the present study, we investigated immunophenotypical changes of KB-D8 tumor cells and tumor-associated macrophages (TAMs) appearing in relation to tumor development in syngeneic F344 rats. A number of neoplastic cells in 0.5-cm-diameter KB-D8 tumors showed immunoreactions to OX6 (specific for rat antigen-presenting cells), ED1 (for rat exudate macrophages), and ED2 (for rat resident macrophages), and 72% and 11% of the OX6+ cells were double-immunostained with ED1 and ED2, respectively. Interestingly, the immunoreactions to these antibodies were gradually reduced with increasing size of KB-D8 tumors of 1-, 2-, and 3-cm diameter. These findings indicated that immunophenotypes of dendritic cell-derived sarcomas may be changeable depending on microenvironmental conditions in vivo. Many TAMs seen outside KB-D8 tumors reacted to OX6, ED1, and ED2; the numbers of TAMs immunopositive for these antibodies also decreased as the tumor grew. Similarly, the earlier temporary increase and subsequent gradual decrease in ED2+ and OX6+ cell numbers were observed in the spleen and liver of KB-D8-bearing rats. The reverse-transcription polymerase chain reaction showed mRNA expressions of granulocyte/macrophagecolony stimulating factor, monocyte-chemoattractant protein-1, and osteopontin in KB-D8 tumor tissues. Although the functional roles (biphasic roles: suppressing or promoting) of these factors should be investigated further in relation to tumor development, the factors might be partially responsible for the TAM reactions. KB-D8 would be a useful experimental model to investigate the biological characteristics of dendritic cell sarcomas and tumor immunology in the host.

Participation of Functionally Different Macrophage Populations and Monocyte Chemoattractant Protein-1 in Early Stages of Thioacetamide-induced Rat Hepatic Injury

“…The double-immunohistochemical staining was carried out according to methods described previously (Kawashima et al 2003). Tissue sections of livers were prepared from three rats at each examination point.…”

“…The cDNA was amplified by the PCR with Taq DNA polymerase and each of the specific primers for rat MCP-1, TGF-b1, and b-actin (control). The following conditions were used for the amplification: for MCP-1 (Kawashima et al 2003), after five minutes of denaturation at 94 C, twenty-seven cycles of fifteen seconds of denaturation at 94 C, thirty seconds of annealing at 53 C, and thirty seconds of extension at 72 C; for TGF-b1 (Ide et al 2005), after five minutes of denaturation at 94 C, 25 cycles of thirty seconds of denaturation at 94 C, one minute of annealing at 60 C, and thirty seconds of extension at 72 C; and for b-actin (Ide et al 2005), after five minutes of denaturation at 94 C, twenty cycles of thirty seconds of denaturation at 94 C, thirty seconds of annealing at 59 C, and thirty seconds of extension at 72 C. The oligonucleotides used for PCR were as follows: MCP-1 sense primer 5 0 -ATG-CAGGTCTCTGTCACG-3 0 and antisense primer 5 0 -CTAGTTCTCTGTCATACT-3 0 (Kawashima et al 2003); TGF-b1 sense primer 5 0 -CACCATCCATGACATGAACC-3 0 and antisense primer 5 0 -GTTGGACAACTGCTCCACCT-3 0 (Ide et al 2005); and b-actin sense primer 5 0 -TAAAGACCTC-TATGCCAACAC-3 0 and antisense primer 5 0 -CTCCTGC TTGCTGATCCACAT-3 0 (Ide et al 2005). The polymerase chain reaction (PCR) products were electrophoresed in 1% agarose gel, and DNA was stained with ethidium bromide on the gel.…”

Increased glial‐derived neurotrophic factor in the small intestine of rats infected with the tapeworm, Hymenolepis diminuta

“…Our Astra blue histochemistry and double immunostaining studies revealed that GDNF reactivity was primarily associated with the increasing macrophage population, but not the mast cells, of the jejunal and ileal mucosa of both uninfected and tapeworm infected rats. Because of the very low intensity and diffuseness of the cytoplasmic staining by ED2 antibody and the documented recognition by this antibody of cytoplasmic and surface membrane antigens of most tissue macrophages (Dijkstra et al 1985a,b;Kawashima et al 2003), the clear perinuclear staining served as our marker of ED2 positive (ED2+) cells for identification, localization and census of macrophages.…”

“…Because of the very low intensity and diffuseness of the cytoplasmic staining by ED2 antibody and the documented recognition by this antibody of cytoplasmic and surface membrane antigens of most tissue macrophages (Dijkstra et al. 1985a,b; Kawashima et al. 2003), the clear perinuclear staining served as our marker of ED2 positive (ED2+) cells for identification, localization and census of macrophages.…”

Immunophototherapy Using PDT Combined with Rapid Intratumoral Dendritic Cell Injection

“…Cells were fed 50% media replacement containing replacement cytokines every 3 days. DCs were evaluated for morphology and cell surface phenotype by flow cytometry using appropriately fluorochrome labeled antibodies to confirm DC phenotype (27–29) prior to use. Our DC preparations routinely exhibit high level staining for CD54, and MHC Class II, positive staining for CD11c, CD80 and CD86, low level positive staining for CD40 and OX62 with other lymphocyte lineage markers being negative and other shared markers with macrophages being positive, e.g.…”