vironment, we performed IHC studies of 53 DLBCL, 20 FL and 12 lymph node hyperplasia, using CD68 24211709 and CD163 monoclonal antibodies. Counting of these macrophages was conducted in accordance with a previous report. Briefly, macrophage counts were estimated 5 times in high-power fields. Mean counts were then determined and used for statistical analysis. In DLBCL, we counted whole macrophages in samples. In FL and RLH controls, we counted intrafollicular macrophages, as a previous result showed that the number of intrafollicular CD68-positive macrophages reflects prognosis of patients with FL. Only areas containing tumors were analyzed for the IHC study, and areas containing only necrosis and fibrosis were excluded. Neutrophils were also excluded. Differences in the number of macrophages in each disease and the relationship between levels of sIL-2R and number of macrophages were evaluated by Mann-Whitney U test and Spearman’s rank correlation coefficient, respectively. Diversity of CD25 expression in lymphoma cells sIL-2R in B-Cell Lymphomas lymphomas. Thus, we hypothesized that some proteinases produced by tumor cells or bystanders cleave IL-2Ra chains. Interestingly, MMP-9 is reported to have the ability to cleave IL2Ra chains. First, we investigated whether IL-2Ra chains on lymphoma cells are cleaved by MMP-9. We were unable to obtain B-cell lymphoma cell lines that were positive for CD25; therefore an ATL cell line was used in this analysis. MT4 cells did not express endogenous MMP-9, and other Celgosivir site CD25-positive cell lines express endogenous MMP-9. We analyzed expression of CD25 by flow cytometry after 6 h of incubation of MT4 with recombinant MMP-9 in FCS-free medium. Treatment with 1 mg/ml rMMP-9 partially decreased expression of CD25, and treatment 12642398 with 3 mg/ml rMMP-9 markedly decreased expression of CD25 in almost all cells. Therefore, MMP-9 is able to cleave IL-2Ra chains depending on its concentration. Subsequently, MT4 cells 
were treated with 400 ng/ml rMMP-9 with or without MMP-9 inhibitor in FCS-free medium. After 6 h of treatment, we then measured sIL-2R in the supernatants of MT4 cells. Levels of sIL2R in supernatants increased with rMMP-9 treatment, but decreased with MMP-9 inhibitor treatment as compared to those of sIL-2R treated with rMMP-9 groups. However, sIL2R was also detected in the supernatants of control groups, which did not express endogenous MMP-9. This raised the possibility that factors in addition to MMP-9 are involved in the cleavage of sIL-2R. Collectively, we confirmed that MMP-9 plays an important role in the cleavage of IL-2Ra chains. Levels of sIL-2R and MMP-9 in serum As it has been demonstrated that MMP-9 cleaves IL-2Ra chains, we then analyzed whether levels of sIL-2R are correlated with those of MMP-9 in patients with DLBCL and FL. We analyzed patient plasma for measurement of MMP-9 as neutrophils and platelets produce their own MMP-9 during the procedure for serum collection. We analyzed levels of sIL-2R and MMP-9 in untreated patients . The average levels of sIL-2R were 2228 U/ml and 2775 U/ml in DLBCL and FL, respectively, and the average concentrations of MMP-9 were 47.2 ng/ml and 34.1 ng/ml in DLBCL and FL, respectively. Correlations between levels of sIL-2R and MMP-9 were evaluated using Spearman’s rank correlation coefficient. In FL, there was a positive correlation, but not in DLBCL . Next, we purified B-cells from lymph nodes of DLBCL, FL and RLH using CD19 microbeads. Cells were cultured in FCS
