TGF-beta1 induces bone marrow reticulin fibrosis in hairy cell leukemia

Abstract

The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-beta1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-beta1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-beta1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-beta1. Active TGF-beta1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-beta1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti-TGF-beta1 antibodies. These results show, for the first time to our knowledge, that TGF-beta1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.

Figures

Figure 1

Levels of TGF-β1 in BMP, serum, and PBP. TGF-β1 was measured by immunoassays before and after acidification to detect active (A) and total (B) TGF-β1. The levels of active and total TGF-β1 were significantly higher in HCL patients (n = 13) than in HDs (n = 10) and B-CLL patients (n = 5). *P < 0.01; **P < 0.001. (C) High expression of TGF-β1 mRNA in PBMCs of HCL patients (lanes 7–12) in comparison with HDs (lanes 1–6). (D) Overexpression of TGF-β1 mRNA in BMMCs of three HCL patients and spleen (spl.) cells of two HCL patients compared with BMMCs of three HDs and PBMCs (lanes 9 and 10) and BMMCs (lanes 11 and 12) of B-CLL patients.

Figure 2

TGF-β1 production by PBMCs and intracellular localization in normal B cells and HCs. (A) Cells were cultured for 48 hours and secreted TGF-β1 (active and total) was measured by ELISA. Significantly higher concentrations of active and total TGF-β1 were detected in supernatants of PBMCs of HCL patients as compared with HD and B-CLL cells. (B–E) Immunofluorescence staining using anti–TGF-β1 (TB21) antibody was performed on purified HCs, normal B cells, and B-CLL cells (>85% leukemic cells). A strong staining for TGF-β1 was found in HCs (B) in comparison with normal B cells (C) and B-CLL cells (D). (E) Nonimmune mouse IgG1 antibody was used as a negative control. A representative of four experiments is demonstrated. Original magnification, ×400.

Figure 3

Secreted and intracellular TGF-β1 in BM cells. (A) BMMCs of HCL patients produced significantly higher amounts of TGF-β1 throughout the incubation than BMMCs of HDs. (B) The amounts of TGF-β1 secreted by BMSCs and BMFs of HCL patients were comparable to the amounts secreted by HD cells. (C and D) Double immunofluorescence staining of BM sections of an HCL patient (representative of four patients tested) shows the colocalization of CD22 (an HC marker) (C) and TGF-β1 (D). (E and F) Cytospin preparation of BMMCs (>90% HCs) double-stained with antibody against CD103 (B-ly7, a specific HC marker) and against TGF-β1 (E and F), confirming the presence of TGF-β1 in the HCs. Original magnification, ×100 (C and D) and ×400 (E and F).

Figure 4

Correlation among TGF-β1 concentrations, grades of BM fibrosis, and the percentage of HCs in the BM. The concentrations of active and latent TGF-β1 in BMP of HCL patients (n = 10) correlate significantly with grades of BM fibrosis (A and B) and with percentage of HCs in BM (C and D). Serum levels of PIIINP significantly correlate with grades of BM fibrosis (E) and with active TGF-β1 in BMP (F). The gray symbols represent the mean value of TGF-β1 in BM (A–D) and PIIINP in serum (E and F) of ten HDs.

Figure 5

In vitro production of collagen and reticulin fibers by BMFs and the effect of TGF-β1. Confluent stationary fibroblast cultures (4–6 weeks) were performed, mature collagen fibers were visualized by Masson’s trichrome staining (A–E), and reticulin fibers were stained by Gomori’s silver impregnation technique (F–J). Under basal conditions, BMFs of HCL patients produced more collagen and reticulin fibers (B and G) than did fibroblasts of HDs (A and F). TGF-β1 (5 ng/ml) increased the number and thickness of collagen and reticulin fibers (C and H), while anti–TGF-β1 antibody significantly decreased fiber deposition (D and I). Control antibody had no effect (E and J). Original magnification, ×600.

Figure 6

Induction of type I and type III procollagen synthesis by BMP of HCL patients. (A–H) BMFs of three HCL patients were cultured and supplemented with 10% BMP of HDs or HCL patients alone or in the presence of neutralizing anti–TGF-β1 antibodies. A representative of three experiments is demonstrated. Type I and type III procollagen was detected mainly intracellularly in the presence of HD BMP (A and E) and was significantly increased in response to HCL BMP (B and F). HCL BMP dramatically increased extracellular deposition of type III procollagen. The effect of BMP was abolished by anti–TGF-β1 antibody (C and G). Control antibody had no effect (D and H). Original magnification, ×400. (I) The effect of BMP on mRNA expression of type 1 (α1) and type III (α1) procollagen in BMFs of three patients with HCL was also investigated, and a representative experiment is shown. BMP of HCL patients (n = 3) had a stronger enhancing effect on the mRNA expression of both types of procollagen (lanes 4–6) than BMP of three HDs (lanes 1–3). The effect of HCL BMP was also abolished by anti–TGF-β1 antibody (lanes 7–9), while the control antibody had no effect (lanes 10–12).

Figure 7

Interaction between HCs and BMFs in vitro. Purified HCs or B cells were cultured on top of BMFs in the absence or presence of anti–TGF-β1 antibody. After 24 hours of incubation, immunofluorescence was performed, and a representative of four experiments is demonstrated. (A) An intense immunoreactivity for TGF-β1 (red fluorescence) was found intracellularly in the HCs (large arrows) and on the matrix produced by the fibroblasts (small arrows). Green fluorescence demonstrates intracellular type III procollagen in the fibroblasts and its extracellular deposition. (B and C) Anti–TGF-β1 antibody significantly inhibited the synthesis of type III procollagen, formation of fibrillar matrix, and deposition of TGF-β1 on this matrix (B), while control antibody had no effect (C). (D) Coculture of normal B cells and BMFs showed a weak TGF-β1 immunoreactivity in B cells (arrow) and absence of extracellular deposition of TGF-β1 and fibrillar matrix. Original magnification, ×400.