To ver ify this suggestion, PBMCs of six HCL patients, six HDs, and five B CLL patients were cultured for 48 hours, and the concentrations of TGF 1 protein in supernatants was measured by ELISA. As illus trated in Figure 2A, PBMCs of HCL patients produced significant ly higher amounts of TGF 1 proteins as com pared with those of HDs, While total TGF 1 produced by PBMCs of HCL patients was fourfold higher, active TGF 1 in HCL patients was 42 fold higher than in HDs, Similarly, the amounts of TGF 1 produced by HCL cells were significantly higher than those produced by B CLL cells. It is important to note that the amount of active TGF 1 pro duced by B CLL cells was similar to that produced by PBMCs of nor mal persons.
To investigate the contribution of HCs to the produc tion of TGF 1, immunofluorescence staining for intracellular TGF 1 was performed in purified HCs from peripheral blood of four HCL patients and compared with normal B cells obtained from four HDs selelck kinase inhibitor and PBMCs of four patients with B CLL, A representative experiment is shown in Figure 2, where HCs show very intense cytoplasmic staining for TGF 1 as com pared with normal B cells and B CLL cells, Furthermore, the purified HCs and B cells were cultured for 48 hours, and TGF 1 concentrations in culture supernatants were measured by ELISA. Significantly higher amounts of active and total TGF 1 were produced by HCs in comparison with normal B cells, These data indicate that the HCs are more efficient in producing TGF 1, particularly in its active form, than are normal B cells and B CLL cells and that they represent a major source for TGF 1 in HCL patients. We therefore focused our inves tigations on cells from HCL patients and HDs. Source of TGF 1 in BM.
To identify the source selleck chemicals of the increased TGF 1 proteins in BM of patients with HCL, a set of experiments was performed using different cellular components of BM, including TGF 1 producing cells in BM of HCL patients, BM sections and aspirates were subjected to double immunofluorescence staining using anti TGF 1 antibody in combination with anti CD22, a marker for HCs,

As demonstrated in Figure 3, C and D, a very intense staining for TGF 1 was found in the majority of HCs, TGF 1 immunoreactivity was also observed in the intercellular space. Furthermore, cytospin preparations of BMMCs were double stained with anti TGF 1 and with anti B ly7 antibodies, which recognize CD103, a specific HC marker, As illustrated in Figure 3, E and F, TGF 1 was also found to be highly expressed specifically in the HCs, Collectively, these results confirm that the HCs represent a major source for TGF 1 within the hematopoietic cells and are thus responsible for the elevated levels of TGF 1 in BM and peripheral blood of HCL patients.