Transforming growth factor beta-1 (TGF-β1) released by an Epstein-Barr virus (EBV) positive spontaneous lymphoblastoid cell line from a patient with Kostmann's congenital neutropenia inhibits the growth of normal committed haemopoietic progenitors in vitro

Summary This study reports the characterization of a spontaneous lymphoblastoid cell line (LCL) raised from the peripheral blood of a patient with Kostmann's congenital neutropenia. The LCL was composed of EBV-infected polyclonal B cells and displayed surface markers and pattern of growth in vitro typical of normal LCLs. The supernatant of the LCL contained a colony inhibiting activity (CIA) that decreased the cloning efficiency of normal committed haemopoietic progenitors and was identified as immunoreactive transforming growth factor β1 (TGF-β1) by neutralization experiments with a specific antiserum. Control studies with a panel of LCLs spontaneously derived from the peripheral blood of patients seropositive for Epstein-Barr virus (EBV) infections showed that 5/30 LCLs produced a CIA. This CIA was not identifiable as TGF-β1 but rather was due to the combined effects of tumour necrosis factor α (TNFα). tumour necrosis factor β (TNFβ) and interferon α (IFNα), that were present in the LCL supernatants. The hypothesis that the B cells latently infected by EBV in vivo and possibly expanded as a consequence of the infection may have contributed to the inhibition of the patient granulopoiesis by releasing TGF-β1 will be discussed.     

Bile acids inhibit tumour necrosis factor α-induced interleukin-8 production in human colon epithelial cells

To clarify the regulatory mechanism of the production of various inflammatory mediators by intestinal epithelial cells, the effect of bile acids (tauroursodeoxycholate, TUDC; taurochenodeoxycholate, TCDC; and taurocholate, TC) on the cytokine-induced production of interleukin (IL)-8 in a human colon epithelial cell line (HT-29) was examined. HT-29 cells were incubated for 24 h in a culture medium containing tumour necrosis factor α (TNFα; 1 ng/mL) and/or interleukin (IL)-1 β (1 ng/mL) in the presence or absence of bile acids. The IL-8 concentration in the medium was measured by an enzyme-linked immunosorbent assay. The binding assay of TNFα was performed using [125I]-TNFα (100 pmol/L). Interleukin-8 production during incubation with TNFα was markedly reduced in the presence of 0.5 and 1 mmol/L TUDC, 0.5 and 1 mmol/L TCDC and 0.5 and 1 mmol/L TC, by 56, 85, 86, 91, 37 and 70%, respectively. The IL-8 production during incubation with IL-1ß was not significantly reduced in the presence of these bile acids. The specific binding of TNFα to cells was inhibited 33, 47, and 14% by 1 mmol/L TUDC, TCDC and TC, respectively. These findings suggest that bile acids inhibit TNFα-induced IL-8 production by the colonic cells. The suppression may be partly due to inhibition of TNFα binding to the cells by bile acids.     

Regulation of Human Monocyte Functions by Acute Ethanol Treatment: Decreased Tumor Necrosis Factorα, Interleukin-lβ and Elevated Interleukin-10, and Transforming Growth Factor-β Production

We and others have previously shown that even acute ethanol exposure has the capacity to modulate immune functions, particularly monocyte functions. Herein, we tested the hypothesis that acute ethanol treatment inhibits inflammatory, while increasing inhibitory cytokine production in human blood monocytes that, in tum, could contribute to the overall immune abnormalities seen after alcohol use. Our data show that in vitro treatment of blood monocytes with a physiologically relevant dose of alcohol (W mM) results in significantly decreased induction of tumor necrosis factor-α (TNFα) and interleukin (IL)-lβ by bacterial stimulation of either Gram-positive [staphylococcal enterotoxin B (SEB), 1 μg/ml of SEB] or Gram-negative [lipopolysaccharide (LPS), 1 μg/ml of LPS] origin both at the protein and mRNA levels. In contrast, acute ethanol treatment induces monocyte production of mediators with immunoinhibitory potential, including transforming growth factor-β and IL-10. We further show that ethanol not only induces monocyte/macrophage (Mø) IL-10 and transforming growth factor-β, but even augments bacterial (both LPS and SEB) stimulation-induced production of both of these cytokines. IL-10 is a potent inhibitor of Mø TNFα production. We found that ethanol-induced elevation in Mø IL-10 levels contributes to the decreased Mø TNFα production to bacterial challenge in eth-anol-exposed Mø. However, mRNA levels for TNFα are downregu-lated as early as 1.5 hr after ethanol treatment, suggesting that ethanol likely has an IL-10 independent, direct effect on early signaling events of TNF α induction.     

Patterns of cytokine expression in AIDS-related non-Hodgkin's lymphoma

The pathogenesis of AIDS-related non-Hodgkin's lymphomas (AIDS-NHL) involves accumulation of genetic lesions, stimulation and selection by antigen, as well as infection by viruses. Deregulation of cytokine loops has also been proposed to contribute to AIDS-NHL development, although data are available only for a limited number of cytokines. In this study we have utilized a panel of AIDS-NHL cell lines to investigate in detail the pattern of tumour expression and production of a wide spectrum of cytokines. The cytokines investigated included interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, TNFα, TNFβ, IFNγ, TGFβ₂, G-CSF, GM-CSF and SCF. The AIDS-NHL cell lines utilized were representative of both AIDS-related Burkitt lymphoma (AIDS-BL) and AIDS-related body cavity-based lymphoma (AIDS-BCBL). Overall, AIDS-NHL were found to produce IL-6, IL-10 and TNFβ, although with different patterns depending upon the biological features of the tumour. Production of high levels of IL10 preferentially associated with Epstein-Barr virus (EBV) positive AIDS-BL and AIDS-BCBL, although lower levels of the cytokine were also detectable among EBV-negative AIDS-BL. Production of IL-6 was restricted to EBV-positive AIDS-BL and AIDS-BCBL, whereas it was absent among EBV-negative AIDS-BL. Production of TNFβ clustered with AIDS-BL, whereas this was absent among AIDS-BCBL. These results define that the pattern of cytokine expression of AIDS-NHL depends upon the biological features of the tumour and may have implications for the pathogenesis of these disorders.     

Inhibition of autophagy abrogates tumour necrosis factor α induced apoptosis in human T-lymphoblastic leukaemic cells

The pattern and the sequence of tumour necrosis factor-α (TNFα) induced cell death in the acute T-lymphoblastic leukaemic cell line CCRF-CEM and its vinblastine-resistant subline CEM/VLB₁₀₀ have been studied. Previously, we found that the CEM/VLB₁₀₀ cell line was more sensitive to TNFα-induced killing than its parental CCRF-CEM cell line. TNFα-induced cell death showed an apoptotic pattern, as detected by agarose electrophoresis, flow cytometry and transmission electron microscopy (TEM). TEM images revealed that autophagy and condensed mitochondria occurred earlier than nuclear fragmentation. The specific inhibitor of autophagy, 3-methyladenine (3MA), inhibited the formation of autophagosomes. TNFα-induced DNA fragmentation and cytolysis were completely inhibited by 10 m M 3MA. Inhibition of the fusion of lysosomes with autophagosomes by asparagine did not block TNFα-induced apoptosis. In addition, amino acid and protein deprivation enhanced TNFα-induced autophagy but not apoptosis. We propose that the early stages of autophagy are required for, but do not necessarily result in, TNFα-induced apoptosis.     

Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: relevance to umbilical cord blood transplantation

Cord blood has been used successfully for stem cell transplantation in several haematological conditions: Fanconi's anaemia, leukaemia and Wiskott-Aldrich syndrome. On account of the low incidence of GVHD observed following cord blood transplantation, it has been suggested that cord blood be used for HLA-matched, or perhaps one or two antigens mismatched, and unrelated stem cell transplantation. Based on an extensive immunophenotype-functional correlation, we determined that cord blood contains mainly immature unprimed T lymphocytes that are predominantly suppressor cells. Recent findings suggest that dysregulated production of cytokines (IL-1, IL-2, TNFα) plays a role in GVHD. We showed that T cells in cord blood express receptors for IL-2, TNFα, but no receptors for IL-1. Similarly, NK cells, one of the effector cells of GVHD, express receptors for TNFα and γIFN but do not express receptors for IL-1, nor IL-2R α-chain (CD25) although IL-2R β-chain is expressed. The potential for activation of T lymphocytes and NK cells therefore exists in the context of bone marrow transplantation. However, the high number of suppressor cells in cord blood most likely modulate the activation of lymphocytes and NK cells thereby minimizing GVHD.     

Regulatory Potential of Ethanol and Retinoic Acid on Human Monocyte Functions

Retinoic acid (RA), a metabolic product of vitamin A, has been shown to affect a variety of immune functions, including monocytes. Monocyte functions and mediator production are also modulated by ethanol exposure. This study demonstrates that therapeutic doses of RA (0.1–10 μM) significantly increase transforming growth factor- β (TGFβ) production both in THP-1, human myelomonocytic cells, and in human peripheral blood monocytes. We have previously reported TGFB induction by ethanol in human M ø. Combination of RA stimulation with acute in vitro ethanol treatment, however, resulted in significantly lower Mø TGFβ production than TGFB levels induced by RA alone ( p < 0.003). Down-regulation of M ø TGFβ production by ethanol was tested at the concentration range of 25–150 mM and occurred both at high and low RA concentrations (10–0.1μM). In contrast to its inhibitory effect on RA-induced M ø TGFβ production, ethanol augmented TGFB production induced by mura-my1 dipeptide (20 μg/ml), suggesting that ethanol can either up- or down-regulate Mø TGFB production, depending on the costimulatory factors. RA also induced a moderate increase in Mø tumor necrosis factor-α (TNFα) production, which was down-regulated by ethanol both at the level of secreted and cell-associated TNFα. In addition to regulation of cytokine production, both RA and ethanol decreased expression of CD4 on THP-1 cells. The degree of inhibition of CD4 expression by RA was more significant than by ethanol, but RA-induced decrease in CD4 expression was not significantly affected by the combined stimulation with ethanol. These results provide further evidence for the immunoregulatory potential of nutritional and dietary factors, such as RA and ethanol, on the immune functions of human monocytes.     

Changes in erythroid progenitor cell and accessory cell compartments in patients with myelodysplastic syndromes during treatment with all-trans retinoic acid and haemopoietic growth factors

Differentiation induction therapy is used in myelodysplastic syndromes (MDS) to improve maturation defects and to restore impaired function of malignant cells. To this end, 18 patients with MDS received either a combination therapy consisting in study 1 of all- trans retinoic acid (ATRA) and granulocyte-colony stimulating factor (G-CSF), or in study 2 of a combination with ATRA, G-CSF, erythropoletin (Epo) and tocopherol. The ANC increased in 19/20 patients in both studies, whereas an increase in haemoglobin concentration, platelet counts or reduction of transfusion requirement was seen in only 8/20 patients, correlating strongly with good BFU-E growth ( P < 0.001). To assess the role of accessory cells in the modulation of the haemopoietic response to treatment, we analysed the capacity of peripheral blood monocytes to secrete cytokines (IL-1β, IL-6, IL-8, TNFα). Secretion of all cytokines was significantly reduced before therapy when compared with healthy controls, but increased during therapy, reaching normal levels for IL-8. These data indicate that a combination therapy with ATRA and cytokines improves impaired cytokine secretion from monocytes and induces a multilineage clinical response in a subgroup of MDS patients characterized by an almost intact erythroid compartment. In contrast, induction of TNFα might be responsible for treatment failure.     

Ethanol Suppresses Endotoxin But Not Platelet Activating Factor-Induced Hypotension and Nitric Oxide

Ethanol (ETOH) inhibits the immune response to endotoxemia. The early stage of endotoxin (LPS)-induced shock is associated with an acute phase cardiovascular depression (APCD). Release of platelet activating factor (PAF) and tumor necrosis factor alpha (TNFa) with upregulation of nitric oxide (NO) production may initiate the APCD. Since ETOH inhibits induction of NO synthase (iNOS) mNRA by LPS, we postulate that ETOH may mask the APCD associated with endotoxemia. To test this, Sprague-Dawley rats (280–320 g, n = 5–6/group) were given LPS [0.75 mg/kg, intravenously (iv)] or PAF (10 to 150 pg/kg, iv) 30 min after administration of sterile saline (PBS), BN-5073 a mixed PAF antagonist (0.50 μg/kg, iv), or ETOH t2.2-5.5 g/kg, intra-peritoneally (ip)]. Cardiovascular parameters and plasma concentrations of nitrate and nitrite (RNI), ETOH, TNFα, and neutrophil (PMN) generation of RNI were measured. LPS and PAF both produced APCD. LPS-induced APCD was associated with tachycardia, elevated plasma TNFα and RNI, and ex wivo generation of RNI by PMNs. ETOH and EN-50730 prevented LPS-induced APCD and increases in RNI and TNFα. ETOH, however, increased the mortality associated with APCD. PAF produced only hypotension, bradycardia and elevated plasma levels of TNFα. ETOH and LNMMA did not affect PAF-induced APCD. EN-50730 inhibited PAF-induced APCD and plasma TNFα. We conclude that 1) ETOH inhibits the APCD and induction of NO characteristic of endotoxemia and 2) ETOH-induced suppression of LPS-mediated APCD may be mediated in part by suppression of release of intracellular PAF. Ethanol may increase the morbidity and mortality of endotoxemia by masking the hypotension and humoral changes characteristic of early endotoxemia thereby delaying appropriate therapy and by diminution of the protective effects of endogenous NO.