B-cell chronic lymphocytic leukaemia cells express and release transforming growth factor-β

Summary Transforming growth factor-β (TGF-β) has been described as a potent inhibitor of various cell types, among others of primitive haematopoietic progenitors in vitro, and as a negative autocrine regulator of normal B lymphocyte growth and differentiation. In the present study we investigated TGF-β gene expression in peripheral blood mononuclear cells (PBMC) and in B cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal controls. Monocyte depleted B-CLL cells expressed constitutively mRNA for TGF-β1 and secreted low amounts of TGF-β activity into the culture medium. Stimulation of cells by phorbol ester noticeably enhanced mRNA levels as well as protein secretion in most cases. TGF-β activity was of the same magnitude as in normal controls. We next analysed TGF-β in highly enriched B lymphocytes from B-CLL (95100% CD19+), and found that TGF-β secretion was up to 3 times higher than in the original PBMC population. It is discussed that B-CLL cells might escape from negative regulation by TGF-β and, on the other hand, inhibit normal haematopoietic cell proliferation and thereby achieve a growth advantage in the haematopoietic tissues.     

Interleukin-13 inhibits interleukin-2-induced proliferation and protects chronic lymphocytic leukemia B cells from in vitro apoptosis

Human interleukin-13 (IL-13) acts at different stages of the normal B- cell maturation pathway with a spectrum of biologic activities overlapping those of IL-4. B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells, arrested at the intermediate stage of their differentiation. In vitro, B-CLL cells exhibit a spontaneous apoptosis regulated by different cytokines. In this report, we show that IL-13 (10 to 200 ng/mL) acts directly on monoclonal B-CLL cells from 12 patients. (1) IL-13 enhances CD23 expression and induces soluble CD23 secretion by B-CLL cells but does not exhibit a growth factor activity. (2) IL-13 inhibits IL-2 responsiveness of B-CLL cells, activated either with IL-2 alone or through crosslinking of lgs or ligation of CD40 antigen. (3) IL-13 protects B-CLL cells from in vitro spontaneous apoptosis. The effects of IL-13 on neoplasic B cells were slightly less than those of IL-4 and occurred independently of the presence of IL-4. The present observations show that IL-13 may exhibit a negative regulatory effect on neoplasic B cells in contrast with that observed in normal B cells, and suggest that IL-13 could be an important factor in the pathogenesis of CLL by preventing the death of monoclonal B cells. Moreover, B-CLL may be an interesting model to study the regulation of the expression of IL-13 receptor and/or signal transduction pathways.     

The two CD23 isoforms display differential regulation in chronic lymphocytic leukaemia

Summary. B lymphocytes from chronic lymphocytic leukaemia (B-CLL) patients express the two CD23 isoforms (type A and B), which differ only in their intracytoplasmic domain. The abnormal regulation of the CD23 antigen in response to IL-4, IFNs α and γ results in CD23 over-expression on B-CLL cells. Our present study shows that the two CD23 isoforms are differentially and abnormally regulated on B-CLL cells. IL-4 selectively up-regulates CD23 type A mRNA in five different B-CLL patients, whereas in normal B cells it enhances CD23 type A and is the most potent inducer of type B. In contrast, phorbol esters (PMA) up-regulate both CD23 isoforms in the malignant B cells and specifically increases type B in normal B cells. We next postulated that cytokines other than IL-4 regulate CD23 B isoform in B-CLL cells and therefore examined the effect of IL-2, IFN-γ and IFN-α. We found that the ability of a given cytokine to induce B-CLL growth (i.e. IL-2 and IFNα) is concurrent with a selective up-regulation of CD23 type B mRNA, whereas lymphokines that have no B cell growth activity (i.e. IL-4 and IFNγ) specifically increase CD23 type A mRNA. We next showed that IL-4 and IFNγ prevent hydrocortisone-induced programmed cell death and that the rescued malignant B cells mainly express CD23 type A. Given that CD23 molecule has been reported to play a role in normal B cell proliferation and survival, it is therefore proposed that in B-CLL cells the expression of CD23 type A may be related to cell viability and that of type B to cell proliferation. These data suggest that the CD23 molecule may contribute to the physiopathology of the disease which is characterized by the accumulation of long-lived and slow-dividing monoclonal B cells.     

Clinical signification of natural killer activity in B-cell chronic lymphocytic leukemia

The natural killer (NK) activity of peripheral blood mononuclear cells (PBMC) and lymphocytes with the capacity to form stable rosettes with neuraminidase-treated sheep red blood cells (E+) was studied in 28 previously untreated patients (11 at stage 0, 10 at stage I and 7 at stages II and III, according to Rai's classification) and 7 treated patients with B-cell chronic lymphocytic leukemia (B-CLL), all of them at stage 0 according to Rai's classification after treatment, and in 15 healthy controls. The mean NK activities of PBMC and E+ lymphocytes from untreated patients were significantly decreased (p less than 0.001) when compared with those of PBMC and E+ lymphocytes, respectively, from healthy controls. However, PBMC and E+ cells from treated patients demonstrated NK activity similar to that of the corresponding cellular populations of controls (p greater than 0.05). Furthermore, there were no significant differences among the NK activities of E+ lymphocytes from untreated B-CLL patients in the different clinical stages 0, I, II and III, according to Rai's classification (p less than 0.05). These results demonstrate that the very low or undetectable levels of NK activity present in PBMC and E+ cell populations from previously untreated patients with B-CLL, regardless of the clinical stage of the disease, can be modified by systemic therapy with alkylating agents. Moreover, the NK activity of PBMC and E+ lymphocytes from some treated patients that have achieved the stage 0 according to Rai's classification after chemotherapy can be found within the range of the lytic activity shown by PBMC and E+ cells from normal donors.     

Cytokine-mediated down-regulation of B cell activity in SLE: effects of interleukin-2 and transforming growth factor-beta

We have recently reported that transforming growth factor-beta (TGF-beta) co-stimulates interleukin-2 (IL-2) activated CD8+ T cells to down-regulate antibody production. In SLE, lymphocyte production of both IL-2 and TGF-beta is decreased. Here we report that a brief treatment of PBMC from SLE patients with IL-2 and TGF-beta can result in marked inhibition of spontaneous polyclonal IgG and autoantibody production. Peripheral blood mononuclear cells (PBMC) from 12 patients with active SLE were exposed to IL-2 with or without TGF-beta for three days, washed and cultured for seven more days. The mean decrease in IgG secretion was 79%. The strongest inhibitory effect was observed in cases with the most marked B cell hyperactivity. Spontaneous production of anti-nucleoprotein (NP) antibodies was observed in four cases and cytokine treatment of PBMC decreased autoantibody production by 50-96%. IL-2 inhibited Ig production by either TGF-beta-dependent or independent mechanisms in individual patients. In a study of anti-CD2 stimulated IgG production in a patient with active SLE, we documented that IL-2 and TGF-beta reversed the enhancing effects of CD8+ T cells on IgG production and induced suppressive activity instead. These results raise the possibility that cytokine-mediated down-regulation of B cell hyperactivity in SLE may have therapeutic potential.     

Production of B-cell growth factor interleukin 2 and gamma interferon by peripheral blood lymphocytes from patients with chronic lymphocytic leukemia of B-cell type

Chronic lymphocytic leukemia of B-cell type (B-CLL) is a malignant disease characterized by monoclonal proliferation of small lymphocytes of B-cell origin, usually associated with suppression of polyclonal B-cell activation (i.e., proliferation and differentiation). Normal human B-cell proliferation is controlled by different T-cell-derived lymphokines, including interleukin 2 (IL2) and gamma interferon (gamma-IFN), that account for the majority of the B-cell growth factor (BCGF) activity produced by mitogen-activated peripheral blood mononuclear cells (PBMCs). We have previously shown an increased and dysregulated secretion of IL2 in peripheral blood from patients with B-CLL. BCGF, IL2, and gamma-IFN productions by phytohemagglutinin (PHA)-stimulated PBMCs were investigated in 13 patients with active untreated B-CLL and 11 healthy donors. B-CLL PBMCs produced a significant amount of BCGF (6 U/ml) despite the low percentage of T cells (10%) associated with this disease as compared with that found in healthy donors (61%). BCGF production in normal controls and B-CLL patients was tripled after irradiation of PBMCs or addition of indomethacin. gamma-IFN secretion in B-CLL patients was decreased when compared with normal controls. Therefore, when gamma-IFN was calculated per fixed number of T cells, production was significantly higher in B-CLL patients than in normal controls, showing a dilution of the productive cells. This study suggests that T cells from B-CLL patients are functional in terms of BCGF production despite their decreased percentage and abnormalities in surface markers.     

Transforming growth factor-beta1: differential effects on multiple myeloma versus normal B cells

Interleukin-6 (IL-6), a product of bone marrow stromal cells (BMSCs), is a growth factor for multiple myeloma (MM) cells. Transforming growth factor-beta1 (TGF-beta1) is also produced by BMSCs and can regulate IL- 6 secretion by several tissues, including BMSCs. The present study was designed to characterize in vitro tumor growth regulation by TGF-beta1 in MM. Sorted CD38+CD45RA- MM cells secreted significantly more TGF- beta1 (8.2 +/- 2.0 ng/mL) than peripheral blood mononuclear cells (P < .001), splenic B cells (P < .001), and CD40 ligand (CD40L) pretreated B cells (P < .05). TGF-beta1 secretion by MM-BMMCs (3.8 +/- 0.9 ng/mL) was significantly greater than by N-BMMCs (1.2 +/- 0.1 ng/mL, P < .001). MM-BMSCs also secreted significantly more TGF-beta1 (6.6 +/- 2.5 ng/mL, n = 11) than N-BMSCs (4.4 +/- 0.6 ng/mL, P < .02, n = 10) and N- BMSC lines (3.9 +/- 0.2 ng/mL, P < .02, n = 6). TGF-beta1 secretion was correlated with IL-6 secretion in MM-BMSCs. Anti-TGF-beta1 monoclonal antibody both blocked IL-6 secretion by BMSCs and inhibited the increments in IL-6 secretion by BMSCs induced by MM cell adhesion. Moreover, exogenous TGF-beta1 upregulated IL-6 secretion by MM-BMSCs, normal BMSCs, and CD38+ CD45RA- MM cells, as well as tumor cell proliferation. This is in contrast to the inhibitory effect of TGF- beta1 on proliferation and Ig secretion of normal splenic B cells. Finally, retinoblastoma proteins (pRB) are constitutively phosphorylated in MM cells; TGF-beta1 either did not alter or increased pRB phosphorylation. pRB are dephosphorylated in splenic B cells and phosphorylated in CD40L triggered B cells in contrast to its effects on MM cells, TGF-beta1 decreased phosphorylation of pRB in CD40L treated B cells. These results suggest that TGF-beta1 is produced in MM by both tumor cells and BMSCs, with related tumore cell growth. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF-beta1 on normal B-cell proliferation and Ig secretion.     

Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non-neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high-performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.     

Expression of TGF-beta gene in adult T cell leukemia

Peripheral mononuclear cells from adult T cell leukemia (ATL) patients were analyzed in comparison with other types of leukemia cells, for the expression of transforming growth factor-beta (TGF-beta) mRNA, for the presence of TGF-beta activity (colony stimulating activity for normal rat kidney fibroblasts [NRK]) in conditioned medium and for their susceptibility to exogenous TGF-beta. Highly elevated TGF-beta mRNA levels were observed in all five ATL cell samples tested; however, in three acute myelogenous leukemia (AML) samples, in one acute lymphatic leukemia (ALL), and one chronic myelogenous leukemia (CML), TGF-beta expression was relatively lower. In normal peripheral mononuclear cells TGF-beta mRNA was weakly detectable. Colony stimulating activity for NRK found in the conditioned medium from ATL cells as well as other leukemia cells correlated well with the levels of TGF-beta mRNA expression. In all three ATL samples tested, stimulation of 3H- thymidine uptake by purified TGF-beta from platelets was apparent. These results suggest that ATL cells are secreting active TGF-beta in a relatively high amount, as compared with other leukemia cells, and may proliferate in response to the factor via an autocrine manner. Furthermore, considering that TGF-beta stimulates bone resorption, we can speculate that the relatively high amount of TGF-beta in ATL cells contributes to the hypercalcemia frequently seen in ATL patients.     

Terminal differentiation of spontaneous rheumatoid factor–secreting B cells from rheumatoid arthritis patients depends on endogenous interleukin-1 0

Objective. The presence of serum rheumatoid factor (RF) and spontaneous RF-secreting B cells is a common feature in most patients with rheumatoid arthritis (RA). This study analyzed the cytokine(s) that controls the final maturation of B cells capable of spontaneous IgM-RF secretion in vitro. Methods. RA patients' peripheral blood mononuclear cells (PBMC), as well as adherent and nonadherent cell fractions, were cultured, and spontaneous IgM-RF and interleukin-1 0 (IL-1 0) secretion were determined by enzyme-linked immunosorbent assay. Results. The RF+ RA PBMC, but not PBMC from RF– RA patients or healthy controls, actively produced IgM-RF in a linear manner for 14 days. This activity depended on the presence of fetal calf serum and did not require cellular DNA synthesis. Spontaneous IgM-RF secretion depended on IL-1 0, as deduced from the following findings: 1) IL-1 0, but not a variety of cytokines including IL-6, restored missing IgM-RF secretion by PBMC in serum-free supplemented cultures; 2) the addition of anti–IL-1 0, but not anti–IL-6, blocking antibodies inhibited PBMC IgM-RF secretion, and this effect could be reversed by exogenous IL-1 0; and 3) RA PBMC actively produced IL-1 0 in vitro. The cells responsible for endogenous IL-1 0 production were found in the adherent cell fraction. Finally, IL-1 0 induced IgM-RF, but not total IgM, secretion by RA PBMC. Conclusion. In patients with RA, circulating B cells capable of spontaneous IgM-RF secretion require IL-1 0 production by adherent cells to reach terminal maturation.     

Transforming growth factor beta 1 regulates production of acute-phase proteins.

We explored the possible role of transforming growth factor beta 1 (TGF-beta), a cytokine that appears to be an important modulator of inflammation and tissue repair, in regulation of human plasma protein synthesis during the acute-phase response. In Hep 3B cells, TGF-beta led to increased secretion of the positive acute-phase proteins alpha 1-protease inhibitor and alpha 1-antichymotrypsin and decreased secretion of the negative acute-phase protein albumin. In Hep G2 cells, after incubation with TGF-beta, the same changes in secretion of alpha 1-protease inhibitor, alpha 1-antichymotrypsin, and albumin were observed, as well as decreased secretion of both the negative acute-phase protein alpha-fetoprotein and the positive acute-phase protein fibrinogen. In addition, TGF-beta modulated the effects of interleukin 6; these cytokines, in combination, were additive in inducing synthesis and secretion of alpha 1-protease inhibitor and alpha 1-antichymotrypsin and in decreasing secretion of albumin and alpha-fetoprotein. TGF-beta inhibited the induction of fibrinogen caused by interleukin 6. The effects on alpha 1-protease inhibitor were confirmed by metabolic labeling in Hep 3B cells and by demonstrating increased accumulation of specific mRNA in Hep G2 cells, and the effects on fibrinogen were confirmed in Hep 3B cells by studies of mRNA for the alpha chain of fibrinogen. TGF-beta had no effect on haptoglobin or alpha 1-acid glycoprotein secretion, either directly or in the presence of interleukin 6, which is capable of inducing these proteins. These studies demonstrate that TGF-beta can affect hepatic synthesis and secretion of a subset of acute-phase proteins, both directly and by modulating the effect of interleukin 6. The affected group of plasma proteins is distinct from those affected by other recognized acute-phase protein-inducing cytokines. These findings support the view that combinations of cytokines mediate the response of the hepatocyte to inflammatory stimuli.     

Evidence that large granular lymphocytes from B-CLL patients with hypogammaglobulinemia down-regulate B-cell immunoglobulin synthesis [published erratum appears in Blood 1989 Jun;73(8):2232]

B-chronic lymphocytic leukemia (CLL) patients frequently suffer from moderate to severe hypogammaglobulinemia. This complication is a serious cause of morbidity and mortality in this disorder. There is recent evidence that natural killer (NK) cells modulate B-cell immunoglobin (Ig) synthesis/secretion. The authors therefore evaluated the circulating NK cells from B-CLL patients on their ability to regulate mitogen-induced B-cell Ig synthesis. Blood, NK cells (CD16+, CD3-) from three B-CLL patients with hypogammaglobulinemia were able to clearly down-regulate the pokeweed mitogen (PWM)-induced-B-cell Ig secretion. In contrast, CD16+, CD3- cells from age-sex-matched controls or B-CLL patients with normal Ig were either nonregulatory or enhanced mitogen-induced B-cell Ig secretion. An alternative explanation for hypogammaglobulinemia in B-CLL patients is the immunomodulation of B- cell Ig production/secretion by CD16+, CD3- blood cells.     

Interleukin 2 production in bone marrow of normal individuals and patients associated with B-cell chronic lymphocytic leukaemia

T-cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) have abnormal T4/T8 ratios and functions. Previously, we demonstrated that peripheral blood (PB) mononuclear cells from B-CLL patients secrete significant amounts of interleukin 2 (IL2) with an apparent dysregulation of accessory cells controlling this production. In this study, IL2 production was investigated in PB and in bone marrow (BM) from patients with previously untreated B-CLL, mostly in early stages of the disease, and compared to normal donors. A significant secretion was observed in both PB and BM from B-CLL patients after stimulation by phytohaemagglutinin (PHA), with lower amounts in patients with nodular involvement of BM, as compared to interstitial or diffuse involvements. To explore the role of accessory cells in controlling IL2 production, we added phorbol ester or indomethacin to the culture system or irradiated the cells before culture. Phorbol ester significantly increased the IL2 secretion in both the PB and the BM of B-CLL patients. The irradiation or the addition of indomethacin did not enhance the IL2 production in PB from B-CLL patients. However, IL2 secretion increased in the BM cells from B-CLL patients after addition of indomethacin or prior irradiation, in a similar way to that observed in PB and BM of normal controls, suggesting an apparent normal control of the IL2 production in BM from B-CLL patients. In normal controls, we demonstrated that IL2 secretion per T-cell from BM was 5.4-fold greater than that from normal PB, suggesting a very efficient role of accessory cells controlling IL2 production in normal BM.     

Distinct characteristics of lymphokine-activated killer (LAK) cells derived from patients with B-cell chronic lymphocytic leukemia (B-CLL). A factor in B-CLL serum promotes natural killer cell-like LAK cell growth

We show that lymphokine-activated killer (LAK) cell precursors derived from patients with B-cell chronic lymphocytic leukemia (B-CLL) and cultured in the presence of recombinant interleukin-2 and normal human serum (NHS), develop into primarily NK cell-like (CD 57+) LAK cells, whereas identically prepared LAK cell precursors from normal subjects develop into mainly T cell-like (CD 3+, CD 8+) LAK cells. B-CLL LAK cells exhibited greater proliferative capacity than did normal LAK cells. When normal LAK cells were grown in B-CLL serum instead of NHS, their proliferation increased; NK cell levels also increased to those found in B-CLL LAK cells, suggesting that B-CLL serum contains a factor that promotes NK cell-like growth, LAK cells derived from normal or B-CLL patients demonstrated similar lytic activity toward K562 and Raji cells. Growth in B-CLL serum did not reduce their lytic potential. Thus, the altered phenotype and growth exhibited by B-CLL LAK cells and normal LAK cells grown in B-CLL serum does not lead to abnormalities in their cytolytic functions. We propose instead that the predominance of NK-like cells in B-CLL LAK cell populations and the presence of an NK cell-like growth factor in B-CLL serum reflect abnormalities related to NK cell-mediated B-cell regulation; ie, either inhibition of normal B-cell growth and/or growth stimulation of the leukemic clone in B-CLL.     

Adenosine 3',5'-monophosphate and phorbol ester induce transforming growth factor-beta 1 messenger ribonucleic acid levels in choriocarcinoma cells.

Transforming growth factor-beta 1 (TGF-beta 1), a regulator of growth and differentiation of many cell types, has previously been purified from the human placenta, and the messenger (m) RNA is abundantly expressed there. We found that the approximate 2.5-kilobase TGF-beta 1 mRNA is expressed in JEG-3 human choriocarcinoma cells, which have been widely used as a model system for studying the regulation of trophoblast hormone secretion. Cholera toxin (CT) elevates the cellular levels of the second messenger cAMP and increases the secretion of CG and steroids in these cells, thus being a potent inducer of trophoblast-differentiated functions. We show that CT also stimulates TGF-beta 1 mRNA levels in JEG-3 cells in a concentration- and time-dependent manner as studied by Northern and dot blotting. The maximal effect (about 5-fold increase above basal levels) occurs within 12-48 h of induction with a CT concentration of 1.0 ng/ml. The cell-permeable cAMP-analog 8-bromo-cAMP stimulates the accumulation of TGF-beta 1 mRNA in JEG-3 cells as well. Furthermore, this cAMP analog also induces TGF-beta 1 mRNA levels in normal cultured term placental cytotrophoblasts. 12-O-Tetradecanoyl phorbol-13-acetate, an active phorbol ester protein kinase C regulator and inducer of TGF-beta 1 mRNA in many cells, increases TGF-beta 1 mRNA accumulation in JEG-3 cells with a similar time course as cAMP analogs but to a lesser extent. Human HT-1080 fibrosarcoma and A-549 lung carcinoma cells exhibit up-regulation of TGF-beta 1 mRNA in response to TGF-beta 1 itself, but we show that activation of the cAMP-dependent pathway does not affect TGF-beta 1 mRNA levels in these cells. Cycloheximide, an inhibitor of protein synthesis, prevents the effect of CT and 12-O-tetradecanoyl phorbol-13-acetate on TGF-beta 1 mRNA expression in JEG-3 cells, suggesting that a protein mediator may be involved in the transduction of their effects. Our finding of a cAMP-dependent induction pathway for TGF-beta 1 mRNA expression in JEG-3 cells provides a new mechanism for the regulation of the synthesis of this ubiquitous growth and differentiation factor and suggests that TGF-beta 1 may have a role in trophoblast differentiation.     

Serum level of transforming growth factor-beta1 (TGF-beta1) and the expression of TGF-beta receptor type II in peripheral blood mononuclear cells in patients with autoimmune hepatitis

BACKGROUND/AIMS: To evaluate the association of the immunosuppressive effects of transforming growth factor-beta1 (TGF-beta1) with abnormalities in immune regulation in autoimmune hepatitis (AIH), we investigated the serum level of TGF-beta1 and expression of TGF-beta receptor type II (TbetaRII) in peripheral blood mononuclear cells (PBMC) in patients with AIH. METHODOLOGY: Twenty-two patients with AIH were included in this study. Serum levels of total TGF-beta1 were determined using a specific enzyme-linked immunosorbent assay (ELISA). The expression levels of TbetaRII mRNA were semi-quantitatively determined by ribonuclease (RNase) protection assay specific for TbetaRII. RESULTS: The mean serum level of TGF-beta1 in patients with AIH (230+/-95 ng/mL) was higher than that of healthy controls (137+/-81 ng/mL, p=0.012). The expression level of TbetaRII mRNA in PBMC obtained from AIH patients (0.131+/-0.046) was lower than that in PBMC of patients with chronic hepatitis C (0.186+/-0.074, p=0.019) and that of healthy subjects (0.188+/-0.060, p=0.013). CONCLUSIONS: The present study of TbetaRII mRNA expression in PBMC from patients with AIH suggested that the decreased expression of TbetaRII might contribute partially to abnormalities in immune regulation observed in patients with AIH, despite concomitant up-regulation of TGF-beta1 production.     

Expression and secretion of type beta transforming growth factor by activated human macrophages.

Alveolar macrophages activated with concanavalin A and peripheral blood monocytes activated with lipopolysaccharide secrete type beta transforming growth factor (TGF-beta). There is minimal TGF-beta secretion in unactivated monocytes, even though TGF-beta mRNA is expressed in these cells at a level similar to that in activated, lipopolysaccharide-treated cultures. U937 lymphoma cells, which have monocytic characteristics, also express mRNA for TGF-beta. Freshly isolated monocytes, both control and lipopolysaccharide-treated, secrete an acid-labile binding protein that inhibits TGF-beta action. We conclude the following: (i) that expression of TGF-beta mRNA is unrelated to monocyte activation, (ii) that secretion of TGF-beta is induced by monocyte activation, and (iii) that cosecretion of TGF-beta and its monocyte/macrophage-derived binding protein may modulate growth factor action. In contrast, monocytic expression of other growth factor genes, such as the B chain of platelet-derived growth factor, is not constitutive and requires activation.     

Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype

OBJECTIVE: CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. METHODS: Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. RESULTS: The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. CONCLUSION: Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B-CLL patients.