Transforming growth factor beta 1 selectively regulates early murine hematopoietic progenitors and inhibits the growth of IL-3-dependent myeloid leukemia cell lines.

Transforming growth factor beta 1 (TGF-beta 1) has been shown to be associated with active centers of hematopoiesis and lymphopoiesis in the developing fetus. Therefore, the effects of TGF-beta 1 on mouse hematopoiesis were studied. TGF-beta 1 is a potent inhibitor of IL-3-induced bone marrow proliferation, but it does not inhibit the proliferation induced by granulocyte/macrophage, colony-stimulating factor (CSF), granulocyte CSF, and erythropoietin (Epo). TGF-beta 1 also inhibits IL-3-induced multipotential colony formation of bone marrow cells in soft agar, which includes early erythroid differentiation, while Epo-induced terminal differentiation is unaffected. In addition, IL-3-induced granulocyte/macrophage colonies were inhibited; however, small clusters of differentiated myeloid cells were consistently seen in cultures containing IL-3 and TGF-beta 1. Thus, TGF-beta 1 selectively inhibits early hematopoietic progenitor growth and differentiation but not more mature progenitors. TGF-beta 1 is also a potent inhibitor of IL-3-dependent and -independent myelomonocytic leukemic cell growth, while the more mature erythroid and macrophage leukemias are insensitive. Therefore, TGF-beta 1 functions as a selective regulator of differentiating normal hematopoietic cells, and suppresses myeloid leukemic cell growth.

The role of human cytomegalovirus in haematological diseases.

Infection with HCMV in healthy individuals generally results in mild or subclinical illness. Pathogenic infections occur predominantly in immunodeficient patients, such as transplant recipients, neonates and patients with AIDS. Primary infection is frequently latent or chronic and PBLs represent sites for virus latency and persistence. HCMV can be recovered from PMNL, monocytes and T-lymphocytes. Although virus-related cases of haematopoietic dysfunction are seen infrequently in infected normal persons, the importance of HCMV as a pathogenic agent in haematopoiesis is dramatically illustrated in the case of patients receiving BMT. Primary or reactivated HCMV infections are a common feature in BMT recipients, enhancing failure of marrow engraftment, GVHD, and many opportunistic infections. HCMV can infect both haematopoietic progenitor cells and stromal elements, identifying the entire haematopoietic system as a target for HCMV dissemination and latency. As a result, lympho- and myelosuppression can be due to both direct inhibition of progenitor cell growth as well as the failure of stem cell self-renewal due to stromal cell dysfunction. HCMV can also exert suppressive effects on immune cell function by direct and indirect mechanisms. These effects can have dire consequences, particularly when a state of immunosuppression already exists, as in the HIV infection. The diverse effects of CMV on the lymphohaematopoietic system are summarized in Figure 1.

Decreased expression of receptors on monocytes from cancer patients.

The expression of crystallizable fragment and complement receptor sites was significantly decreased on the peripheral blood monocytes from 10 patients with advanced carcinoma. In addition the phagocytic activity of these cells was depressed, although no differences in the content of lysosomal enzymes could be demonstrated by staining techniques. These results suggest that the functions of monocytes from such cancer patients may be severely depressed.

The effects of human cytomegalovirus challenge in vitro on subpopulations of T cells from seronegative donors.

Purified T cells from seronegative donors were challenged with human cytomegalovirus (CMV) and assayed for response to the mitogen phytohemagglutinin (PHA) administered at various times after virus challenge. Cells were hyporesponsive to PHA added up to 6 h postinfection, while cells challenged with ultraviolet (UV)-inactivated CMV showed normal PHA responses. T-cell populations showed a complete inversion of helper/suppressor ratios by 6 h of infection, as determined by indirect immunofluorescence using the monoclonal antibodies OKT4 and OKT8. This increased proportion of suppressor cells was functionally demonstrable in an autologous one-way mixed lymphocyte reaction (AMLR).

Preferential suppression of myelopoiesis in normal human bone marrow cells after in vitro challenge with human cytomegalovirus.

The pathogenic effects of human cytomegalovirus (CMV) infection in vitro on hematopoiesis were investigated. Normal human bone marrow cells from both seronegative and seropositive donors were challenged with CMV (Towne or wild-type strain) and tested for their responsiveness to the recombinant hematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF), respectively. Regardless of the serostatus of the donor, infection with CMV resulted in a significant decrease in the proliferation and colony formation of hematopoietic progenitor cells in response to both growth factors, with more pronounced suppression in response to G-CSF being observed. Evaluation of the colony composition revealed a profound decrease in colonies of the granulocytic (CFU-G), or granulocyte-macrophage (CFU-GM) lineages, while suppression of multipotential (CFU-GEMM) and erythroid (BFU-E) colony-forming cells occurred after infection with wild-type but not the laboratory strain of CMV. Although no evidence of productive virus infection could be seen in colony-forming cells, in situ hybridization studies and immunohistochemical staining revealed the presence of CMV-specific mRNA and immediate-early antigens, demonstrating that a small proportion of cells were abortively infected. These studies demonstrate that CMV can infect bone marrow progenitor cells and interfere with normal hematopoiesis in vitro, which may help to explain the hematologic defects seen during acute infections with CMV in vivo.

Hepatitis B virus differentially suppresses myelopoiesis and displays tropism for immature hematopoietic cells.

The hematopoietic cell lines HL-60 and THP-1 were challenged with hepatitis B virus (HBV) in vitro to study interactions between the virus and host cell. Exposure to HBV suppressed the ability of HL-60 cells to differentiate into granulocytes after treatment with retinoic acid (RA) or dimethyl sulfoxide (DMSO), and RA-induced activation of the monocytic cell line THP-1 was also suppressed. Terminal differentiation of both cell lines by phorbol 12-myristate 13-acetate (PMA) was not affected by HBV. The suppressive effect on RA- or DMSO-induced differentiation was unique to HBV, since cell exposure to human cytomegalovirus, another virus that inhibits hematopoiesis, failed to block cellular differentiation. At 5 days postinfection, extracellular viral DNA was detected in immature but not in differentiated cultures and higher levels of core antigen (HBcAg) and surface antigen (HBsAg) were seen in undifferentiated cells than in RA- or PMA-treated cells. In addition, release of HBsAg into the medium was 2 to 12 times greater in untreated cultures than for RA- or PMA-treated cells. Thus, HBV suppresses hematopoiesis by blocking the maturational development of progenitors and selectively infects immature myeloid cells compared with mature end-stage cells.

Transforming growth factor beta selectively inhibits normal and leukemic human bone marrow cell growth in vitro.

The effects of transforming growth factor beta 1 or beta 2 (TGF-beta 1 or -beta 2) on the in vitro proliferation and differentiation of normal and malignant human hematopoietic cells were studied. Both forms of TGF-beta suppressed both the normal cellular proliferation and colony formation induced by recombinant human interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In the presence of GM-CSF or IL-3, optimal concentrations of TGF-beta (400 pmol/L) inhibited colony formation by erythroid (BFU-E), multipotential (CFU-GEMM), and granulocyte-macrophage (CFU-GM) progenitor cells by 90% to 100%, whereas granulocyte or monocyte cluster formation was not inhibited. In contrast, neither form of TGF-beta had any effect on G-CSF-induced hematopoiesis. The suppressive action appeared to be mediated directly by TGF-beta since antiproliferative responses were also observed in accessory cell-depleted bone marrow cells. In contrast to normal bone marrow cells, both GM- and G-CSF-induced proliferation of cells from patients with chronic myelogenous leukemia were suppressed in a dose-dependent manner by TGF-beta. Differential effects of TGF-beta on the proliferation of established leukemic lines were also observed since most cell lines of myelomonocytic nature studied were strongly inhibited where erythroid cell lines were either insensitive or poorly inhibited by TGF-beta. These results suggest that TGF-beta is an important modulator of human hematopoiesis that selectively regulates the growth of less mature hematopoietic cell populations with a high proliferative capacity as opposed to more differentiated cells, which are not affected by TGF-beta.

Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53.

We wished to examine the role of transforming growth factor-beta (TGF-beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF-beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti-Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.

Transforming growth factor-beta 1 enhances the suppression of human hematopoiesis by tumor necrosis factor-alpha or recombinant interferon-alpha.

The effects of transforming growth factor-beta 1 (TGF-beta 1) on human hematopoiesis were evaluated in combination with two other regulatory cytokines, namely, recombinant human tumor necrosis factor-alpha (TNF-alpha) and recombinant human interferon-alpha (rIFN-alpha). Combinations of TNF-alpha and TGF-beta 1 resulted in a synergistic suppression of colony formation by erythroid progenitor cells (BFU-E) and an additive suppression of granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. In addition, TGF-beta 1 synergized with rIFN-alpha to suppress CFU-GM formation, while the combined suppressive effects of both cytokines on CFU-GEMM and BFU-E were additive. When TGF-beta 1 was tested with TNF-alpha or IFN-alpha on granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cells in a 5-day proliferation assay, the antiproliferative effects of TGF-beta 1 and TNF-alpha were additive, while those with TGF-beta 1 and rIFN-alpha were synergistic. A similar pattern was seen in the suppression of the myeloblastic cell line KG-1 where TGF-beta 1 in combination with TNF-alpha resulted in an additive suppression while inhibition by TGF-beta 1 and IFN-alpha was synergistic. These results demonstrate for the first time the cooperative effects between TGF-beta and TNF-alpha and IFN-alpha in the suppression of hematopoietic cell growth, raising the possibility that TGF-beta might be used in concert with TNF-alpha or IFN-alpha in the treatment of various myeloproliferative disorders.

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth.

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.

Infection of human bone marrow stromal cells by hepatitis B virus: implications for viral persistence and the suppression of hematopoiesis.

Suspension cultures of bone marrow cells (BMC) were challenged with hepatitis B virus (HBV) to study interactions between the virus and the nonadherent and adherent BMC populations. Virus-challenged BMC developed an adherent stromal layer that differed in cellular composition from that of mock-infected cultures, showing a threefold increase in cells of the monocyte-macrophage lineage with an accompanying decrease in cells of the granulocytic lineage. Both viral envelope hepatitis B surface and core antigen expression was detected in adherent and nonadherent cell populations up to 10 days after virus challenge, which decreased thereafter. HBV DNA was still detectable in adherent cells 3 weeks after virus challenge, as shown by polymerase chain reaction analysis. These data indicate that HBV can infect not only bone marrow colony-forming cells but also the stromal cell populations involved with the regulation of hematopoiesis in vivo. Such virus-cell interactions could contribute to the immune dysfunction and bone marrow failure occasionally reported for patients with HBV infection as well as acting as an important site for HBV latency and persistence.

A molecular comparison of T lymphocyte populations infiltrating the liver and circulating in the blood of patients with chronic hepatitis B: evidence for antigen-driven selection of a public complementarity-determining region 3 (CDR3) motif.

Despite a large number of T cells infiltrating the liver of patients with chronic hepatitis B, little is known about their complexity or specificity. To characterize the composition of these T cells involved with the pathogenesis of chronic hepatitis B (CHB), we have studied the clonality of VbetaT cell receptor (TCR)-bearing populations in liver tissue by size spectratyping the complementarity-determining region (CDR3) lengths of TCR transcripts. We have also compared the CDR3 profiles of the lymphocytes infiltrating the liver with those circulating in the blood to see whether identical clonotypes may be detected that would indicate a virus-induced expansion in both compartments. Our studies show that in most of the patients examined, the T cell composition of liver infiltrating lymphocytes is highly restricted, with evidence of clonotypic expansions in 4 to 9 TCR Vbeta subfamilies. In contrast, the blood compartment contains an average of 1 to 3 expansions. This pattern is seen irrespective of the patient's viral load or degree of liver pathology. Although the TCR repertoire profiles between the 2 compartments are generally distinct, there is evidence of some T cell subsets being equally distributed between the blood and the liver. Finally, we provide evidence for a putative public binding motif within the CDR3 region with the sequence G-X-S, which may be involved with hepatitis B virus recognition.

Establishment of a cell line from a hepatocellular carcinoma from a patient with hemochromatosis.

We describe the establishment and characterization of a novel hepatoma cell line. This cell line, designated RBHF-1, was established from a hepatocellular carcinoma of a 67-yr-old man with a history of genetic hemochromatosis. At this writing, the cells have been maintained in RPMI-1640 tissue-culture medium and fetal calf serum without any additional supplements for 30 mo. The cells form colonies on soft agar and are not tumorigenic in nude mice. The cell line is polymorphic and displays characteristics of mature hepatocytes by synthesizing albumin, alpha 2-macroglobulin, fibronectin and alpha-fetoprotein. Cytogenetic analysis shows multiple chromosomal aberrations, with a consistent deletion in the long arm and deletions or rearrangements in the short arm of chromosome 1. There is no evidence for hepatitis B or hepatitis C virus infection of the cell line. The cells contain no detectable intracellular iron after staining with Perls' stain. Unlike other hepatoma cell lines, there is no detectable binding of epidermal growth factor to RBHF-1 cells. This is the first cell line to be established from a patient with hemochromatosis, and it provides a potentially important model for the study of hepatocyte transformation in association with iron overload.

A longitudinal analysis of cytotoxic T lymphocyte precursor frequencies to the hepatitis B virus in chronically infected patients.

Individuals with acute hepatitis B virus (HBV) infection characteristically mount a strong, multispecific cytotoxic T lymphocyte (CTL) response that is effective in eradicating virus. In contrast, this response in chronic carriers is usually weak or undetectable. Since it is generally acknowledged that HBV pathogenesis is immune-mediated, the occurrence of episodes of active liver disease in many carriers suggests that these individuals can mount active CTL responses to HBV. To see whether the detection of circulating CTLs is related to these flare episodes, we have determined the CTL precursor (CTLp) frequencies to HLA-A2-restricted viral peptides in seven patients over a 12-24-month period of their disease. Limiting dilution analyses (LDA) were performed longitudinally to five epitopes comprising the viral capsid (HBc), envelope (HBs) and polymerase (pol) proteins. Assays were performed against a mixture of peptides, or against each individual peptide, to measure overall CTL activity and the multispecificity of the responses, respectively. Since two of the patients were treated with recombinant human interleukin-12 (rHuIL-12) at the time, with one individual achieving complete disease remission a year later after being treated with interferon-alpha, we were also able to examine the effects of these cytokines on HBV cytotoxicity. Our results indicate that weak but detectable CTL responses do occur in chronic carriers which are generally associated with disease flares, although CTLps were also seen occasionally during minimal disease activity. The range of specificities varied between individuals and within each individual during the course of the disease. Finally, we also provide evidence that CTL reactivity is stimulated following treatment with certain cytokines, but is dependent on the time of administration.

Transforming growth factor beta directly regulates primitive murine hematopoietic cell proliferation.

We previously reported that transforming growth factor beta (TGF-beta) selectively inhibits colony-stimulating factor-driven hematopoietic progenitor cell growth. We report here that TGF-beta 1 can act directly on hematopoietic progenitors to inhibit the growth of the most primitive progenitors measurable in vitro. Highly enriched populations of hematopoietic progenitor cells were obtained by isolating lineage negative (Lin-), Thy-1-positive (Thy-1+) fresh bone marrow cells, or by isolating cells from interleukin-3 (IL-3) supplemented bone marrow cultures expressing Thy-1 antigen with the fluorescent activated cell sorter. TGF-beta 1 inhibited IL-3-induced Thy-1 expression on Thy-1-negative (Thy-1-) bone marrow cells in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. In addition, TGF-beta 1 inhibited the formation of multipotent and mixed colonies by isolated Thy-1+ cells, while single lineage granulocyte and macrophage colonies were not affected. The growth of Thy-1+ Lin- cells incubated as single cells in Terasaki plates in medium supplemented with IL-3 were inhibited by TGF-beta, demonstrating a direct inhibitory effect. Hematopoietic stem cells, which have a high proliferative potential (HPP) when responding to combinations of growth factors in vitro, have been detected in the bone marrow of normal mice and mice surviving a single injection of 5-fluorouracil. TGF-beta 1 inhibited the growth of all subpopulations of HPP colony forming cells (CFC) in a dose-dependent manner with an ED50 of 5 to 10 pmol/L. Thus, TGF-beta directly inhibits the growth of the most immature hematopoietic cells measurable in vitro.

Growth inhibition of a human lymphoma cell line: induction of a transforming growth factor-beta-mediated autocrine negative loop by phorbol myristate acetate.

Transforming growth factor-beta (TGF-beta) exerts profound inhibitory effects on a number of cell types, including normal B- and T-lymphocytes. In contrast, we have found a number of lymphoid tumor cell lines to be insensitive to the antiproliferative effects of TGF-beta 1 or TGF-beta 2. Binding and cross-linking with radioiodinated TGF-beta 1 demonstrated either low or absent expression of all three TGF-beta receptor species on three B-cell tumor lines, but T-cell and non-T, non-B tumors expressed large numbers of receptors. Treatment of the B-cell lines with phorbol 12-myristate 13-acetate (PMA) induced the expression of TGF-beta receptors and inhibited proliferation in all three lines in a dose- and time-dependent manner. The cell lines constitutively produced TGF-beta mRNA and released small amounts of latent TGF-beta; however, PMA induced the release of active TGF-beta. A neutralizing antibody to TGF-beta was able to reverse the PMA-induced growth inhibition of the malignant lymphoma cell line, RL, and addition of exogenous TGF-beta reversed the effect of the neutralizing antibody. Thus, TGF-beta can inhibit human lymphoma cell growth in vitro through an autocrine mechanism. Some lymphoma cells appear to have escaped from TGF-beta negative regulation by failing to express functional TGF-beta receptors and/or by failing to secrete active TGF-beta receptors and/or by failing to acts to inhibit lymphoma cell growth is by inducing the expression of TGF-beta receptors and the secretion of active TGF-beta, thereby reestablishing an autocrine growth-inhibitory loop.