Hemin-induced erythroid differentiation changes the sensitivity of K562 cells to tumor necrosis factor-alpha

Tumor necrosis factor-alpha (TNF) can inhibit the growth of erythroid progenitors (erythroid colony-forming units [CFU-E] and erythroid burst-forming units [BFU-E]) at picomolar concentrations, but only if added within the first 48 h of culture. These data suggested that cells undergoing erythroid differentiation become resistant to TNF. To test this hypothesis, K562 cells were treated with hemin to induce erythroid differentiation and then tested for their sensitivity to TNF in terms of growth and TNF receptor expression. TNF inhibited the growth of untreated K562 cells, but not hemin-treated K562 cells. Untreated K562 cells expressed TNF receptors, whereas few hemin-treated K562 cells expressed TNF receptors within 24 h of exposure to hemin. These data show that K562 cells induced to differentiate along the erythroid pathway are resistant to TNF because they lack TNF receptors and suggest that the resistance of erythropoietin-treated human bone marrow cells to TNF added after 48 h of culture may also reflect loss of TNF receptors associated with erythroid differentiation.     

The effects of alkyl-lysophospholipids on leukemic cell lines. I. Differential action on two human leukemic cell lines, HL60 and K562

The action of an alkyl-lysophospholipid (ALP), ET180CH3, on clonogenicity, 3H-TdR uptake, and cell numbers was tested in two human leukemic cell lines, HL60 and K562, and short-term human leukemic bone marrow cultures. ALP eliminated clonogenicity in HL60 but not in K562 cultures; 3H-TdR uptake and cell numbers were depressed at low concentrations of ET180CH3 in HL60, but not K562 cultures. The action of the lysophospholipid analog on human leukemic bone marrow short-term cultures at low concentrations was similar to its action on HL60 cultures; clonogenicity and 3H-TdR uptake were depressed, but cell numbers were not significantly affected. The demonstration of differential action of ALP on two cell lines should significantly simplify the investigation of the mechanism of the reported differential action of ET180CH3 on normal and leukemic cell membranes.     

Carbohydrate antigen profiles of human erythroleukemia cell lines HEL and K562

The expression of major carbohydrate antigens carried by polylactosaminyl chains in human erythroleukemia cell lines, K562 and HEL, was investigated by applying monoclonal antibodies recognizing specific carbohydrate determinants. The two cell lines showed common differences in their glycolipid compositions: (1) the presence of significant amounts of ganglio-series glycolipids, which are absent in normal erythrocytes; and (2) a remarkable reduction in the amount of globo-series glycolipids, which are the major glycolipids in normal human erythrocytes. A variety of differences were also detected in the carbohydrate antigens carried by lacto-series glycolipids and glycoproteins having related carbohydrate chains. K562 cells were i+H- X+, with a minor population of I+ cells. HEL cells were I-i+H+X-. The presence of the I+ population in K562 cells is particularly noteworthy, since I-antigen is characteristic of adult mature erythrocytes and is absent in most human leukemic cell lines. Several clones showing I+, I+/-i+/-, or I-i+ specificities were isolated from K562 cells by cloning in either methylcellulose media or limiting dilution, and I+ and I- cells were sorted by FACS fluorometer. HEL cells and these K562 clones provide a useful experimental model for studying the biologic significance and enzymatic control in expression of cell surface polylactosamines.     

Isolation and induction of erythroleukemic cell lines with properties of erythroid progenitor burst-forming cell (BFU-E) and erythroid precursor cell (CFU-E).

We isolated erythroleukemic cell lines arrested at different levels of the erythroid differentiation pathway. One cell line (CB5), established from mice infected with the helper-independent Friend murine leukemia virus (F-MuLV), exhibited properties similar to those of the normal erythroid progenitor burst-forming cell (BFU-E). Six erythroleukemic cell lines, which were established from the anemia-inducing Friend virus complex (FV-A)-infected mice, formed erythroid colonies similar to the erythroid colony-forming precursor cell (CFU-E) after induction with dimethyl sulfoxide or erythropoietin. Three lines that were established from the polycythemia-inducing Friend virus complex (FV-P)-infected mice also formed low proportions (2-5%) of CFU-E-like colonies after induction by these same inducers. These data, together with the earlier findings that F-MuLV induces an increase in the levels of BFU-E and that FV-A or FV-P stimulates enhancement of CFU-E early after infection, indicate that the erythroleukemic cell lines isolated late in the diseases are at the same levels of differentiation as the leukemic cells in the corresponding initial stages. These cell lines with properties of BFU-E and CFU-E can be induced to differentiate in culture and should add to our understanding of the nature of erythroid progenitor cells and their early differentiation programs.     

HEL and K562 cells: analysis of proteins by two-dimensional gel electrophoresis and comparison to erythroid and nonerythroid cells

We have examined the total protein composition of the erythroleukemia cell lines K562 and HEL using high-resolution two-dimensional gel electrophoresis and have compared them with the pattern obtained by normal erythroid and nonerythroid cells. The proteins from the two cell lines, K562 and HEL, gave two-dimensional patterns that were similar to each other and to that of normal lymphocytes or leukemic cell lines. In contrast, normal erythroid precursor cells (BFUe-derived normoblasts) and erythrocytes have a protein profile that is characteristic and significantly different from that of normal lymphocytes or the leukemic cell lines examined. These data suggest a common protein profile in hemopoietic cells at early stages of differentiation (K562, HEL, or other leukemic lines) and in normal lymphocytes. Erythroid cells, in contrast to lymphoid cells, appear to diverge significantly from this common protein profile when differentiation proceeds to the level of morphologically recognizable erythroid cells. Induction of K562 and HEL cells by hemin produces changes in the abundance of several proteins, but fails to change the overall protein profile of the two cell lines.     

Tumor necrosis factor alpha inhibits insulin-like growth factor I-induced hematopoietic cell survival and proliferation

Proinflammatory cytokines, such as TNFalpha and IL-1beta, are both cytostatic and cytotoxic. In contrast, IGF-I promotes proliferation and survival of hematopoietic progenitor cells. In this report, we establish that both the cytostatic and cytotoxic activity of TNFalpha on murine myeloid progenitor cells is only evident in the presence of IGF-I. We first confirmed that IGF-I (100 ng/ml) increases DNA synthesis and reduces apoptosis in murine myeloid progenitor cells induced to die by growth factor withdrawal. TNFalpha inhibits, in a dose-dependent fashion from 0.1 to 10 ng/ml, both activities of IGF-I. TNFalpha activity was not detected in the absence of IGF-I. Another proinflammatory cytokine, IL-1beta, did not inhibit IGF-I-induced activity in murine factor-dependent cell progenitor-1/Mac-1 cells. However, the ability of TNFalpha to impair IGF-I-induced DNA synthesis in human promyeloid cells extends to IL-1beta. Statistically significant inhibition of all these events occurs at very low concentrations of 1 ng/ml or less. These results support the general concept that proinflammatory cytokines impair the actions of hormones on hematopoietic cells, leading to IGF-I receptor resistance.     

Ciprofloxacin inhibits human hematopoietic cell growth: synergism with tumor necrosis factor and interferon

The cytokines tumor necrosis factor (TNF) and interferon (IFN) induce antiproliferative and cytotoxic activity in a variety of cell types. Ciprofloxacin (CFN)--a new fluoroquinolone antibiotic--has also been described, at high concentrations, to suppress hematopoietic cell growth and to affect cytokine production. This study examines the possible relationship between TNF alpha and IFN gamma, as components of host defense mechanisms, and CFN. To investigate the effect of CFN, either alone or combined with TNF or IFN, on normal human hematopoiesis, we examined in vitro changes in hematopoietic progenitor cell growth. We also studied the effect of CFN on human cytokine production by determining TNF, IFN, and colony-stimulating factor (CSF) production by human mononuclear leukocytes (MNC). Granulocyte and monocyte colony formation (granulocyte-macrophage colony-forming cells, GM-CFC) as well as erythroid burst formation (erythroid burst-forming units, BFU-E) were inhibited only by high nontherapeutic levels of CFN. Lower CFN concentrations, however, were inhibitory in the presence of low, noninhibitory concentrations of human recombinant (r)IFN gamma or rTNF alpha. CFN induced a striking dose-dependent increase in IFN gamma production and a decrease in CSF production by mitogen-stimulated MNC. No effect was observed, however, on TNF production by stimulated MNC. The synergistic inhibition of hematopoietic progenitor cell proliferation, achieved by combining low doses of CFN and of antiproliferative cytokines, may explain the occasional case of leukopenia or anemia observed in infected patients receiving CFN. This effect may also indicate the applicability of such a combination against malignant cell growth.     

Antiproliferative effects of natural human tumor necrosis factor-alpha, interferon-alpha, and interferon-gamma on human pancreatic carcinoma cell lines

The antiproliferative effects of natural human tumor necrosis factor-alpha (nHuTNF-alpha), natural human interferon-alpha (nHuIFN-alpha), and natural human interferon-gamma (nHuIFN-gamma) were investigated in human pancreatic carcinoma cell lines. HuP-T3, HuP-T4, and BxPC-3 carcinoma cells exhibited mild growth inhibition after treatment with nHuTNF-alpha. HuP-T3, HuP-T4, MIA-PaCa-2, and BxPC-3 cells exhibited mild or marked growth inhibition after treatment with nHuIFN-alpha. Incubation with nHuIFN-gamma caused marked growth inhibition of HuP-T4 and BxPC-3 cells, whereas HuP-T1, HuP-T3, and MIA-PaCa-2 cells showed only mild growth inhibition with the same dose of nHuIFN-gamma. Combined nHuTNF-alpha and nHuIFN-alpha (1:1) demonstrated marked synergism in comparison with their effects as single agents on HuP-T1, HuP-T3, and MIA-PaCa-2 cells. The combination of nHuTNF-alpha and nHuIFN-gamma (100:1) also demonstrated a marked synergistic effect in comparison with these cytokines alone in four out of five pancreatic carcinoma cell lines (HuP-T1, HuP-T3, HuP-T4, and MIA-PaCa-2 cell lines). The marked increase in efficacy brought about by using combinations of cytokines may enable some improvement in the treatment of pancreatic carcinoma patients in the future.     

Changes of the phenotype of erythroid progenitor cells (BFU-E, CFU-E) and their progenies during early steps of differentiation

Early differentiation processes of human erythroid progenitor cells (BFU-e, CFU-e) have been studied during in vitro proliferation using a panel of monoclonal antibodies with known reactivity on different levels of the erythroid cell line. Two antibodies recognizing structures on BFU-e (VIP-2b, BMA 021), two antibodies reactive with CFU-e and nucleated red cells (5F1, CLB-Ery-3) and one antibody directed against glycophorin A (VIE-G4) were used for this study. Normal human bone marrow cells were induced to proliferation in an erythroid progenitor cell assay and, after different periods of incubation, agar cultures were treated with these antibodies and complement. Thereafter, the remaining erythroid cells were incubated again to continue their proliferation with the same stimulators as before. The changes of the phenotype of BFU-e and CFU-e progenies during in vitro proliferation were determined by the reduction of colony formation in comparison with untreated control cultures. Our results indicate that the loss of HLA-DR antigens and the p45 structure is accompanied by the acquisition of structures recognized by the antibodies 5F1 and CLB-Ery-3. After 5-7 d of incubation BFU-e derived progenies exhibit the same antigenic structure as has been found for CFU-e. Glycophorin A expression could only be demonstrated at a late differentiation stage of the erythroid cell lineage.     

The effects of tumor necrosis factor-alpha on early human hematopoietic progenitor cells treated with 4-hydroperoxycyclophosphamide

We have previously reported that 20 hours' preincubation of human bone marrow cells with interleukin-1 beta (IL-1) can protect early progenitor cells from 4-hydroperoxycyclophosphamide (4-HC) cytotoxicity. Since tumor necrosis factor-alpha (TNF alpha) shares many of the biologic properties of IL-1, we have compared the protective effects of TNF alpha with IL-1 against 4-HC. Incubation of human bone marrow mononuclear cells or an enriched progenitor population for 20 hours with either TNF alpha or IL-1 resulted in the survival of an increased number of single- and mixed-lineage colonies, including replatable blast cell colonies, while only rare colonies were seen in the control group. Antibodies to TNF alpha completely abolished the protection observed with IL-1, while antibodies to IL-1 alpha and IL-1 beta decreased but did not abolish the protection seen with TNF alpha. Combinations of low doses of TNF alpha and IL-1 showed synergy in their protective effects. Furthermore, no protection was observed by IL-1, IL- 1 bone-marrow-conditioned medium (IL-1-BMCM), or TNF alpha for HL-60, K562, KG1, KG1a, and DU.528 leukemic-cell lines or primary acute myelogenous leukemic (AML) blast cells from the lethal effects of 4-HC. In the case of HL-60 and KG1a cell lines, TNF alpha preincubation resulted in increased cytotoxicity. Furthermore, preincubation of a mixture of AML cells and normal bone-marrow cells with IL-1 + TNF alpha before 4-HC resulted in the protection of normal but not leukemic progenitors. These results suggest that TNF alpha is necessary for the protection of normal, early, human hematopoietic progenitors from 4-HC, while IL-1 is not mandatory but will synergize with TNF alpha to offer increased protection. In addition, no protection from 4-HC is observed by TNF alpha, IL-1, or IL-1-BMCM for primary leukemic blast cells or leukemic cell lines.     

Generation of T cells from adult human hematopoietic stem cells and progenitors in a fetal thymic organ culture system: stimulation by tumor necrosis factor-alpha

To investigate the T-lymphopoietic capacity of human adult bone marrow (ABM) hematopoietic progenitor cells, CD34+Lin-, CD34+CD38+, and CD34++CD38- cells were cultured in a severe combined immunodeficient (SCID) mouse fetal thymic organ culture (FTOC). Direct seeding of these progenitors resulted in a moderate to severe cell loss, particularly for the CD34++CD38- cell fraction, and T cells could only be generated from the CD34+Lin- fraction. Preincubation for 36 hours with interleukin-3 (IL-3) and stem cell factor (SCF) led to an improved cell survival and proliferation, although T-cell development was seen only in the CD34+Lin- fraction. Addition of tumor necrosis factor (TNF)-alpha to IL-3 + SCF-supplemented preincubation medium resulted in optimal cell survival, cell proliferation. and T-cell generation of all 3 cell fractions. The TNF-alpha effect resulted in an up-regulation of CD127 (ie, the IL-7 receptor alpha-chain) in a small subset of the CD34+ cells. No evidence could be generated to support the possibility that TNF-alpha inhibits a cell population that suppresses T-cell differentiation. A quantitatively different T-cell generation potency was still seen between the 3 subpopulations: CD34+Lin- (100% success rate) > CD34+CD38+ (66%) > CD34++CD38- (25%). These data contrast with our previous findings using fetal liver and cord blood progenitors, which readily differentiate into T-lymphocytes in FTOC, even without prestimulation with cytokines. Our results demonstrate that adult CD34++CD38- cells, known to contain hematopoietic stem cells, can differentiate into T-lymphocytes and that a significant difference exists in T-lymphopoietic activity of stem cells derived from ontogenetically different sources. (Blood. 2000;95:2806-2812)     

Inhomogeneity of the circulating BFU-E regulation in sickle cell anaemia: accessory cells properties and BFU-E growth factor response pattern

Summary. Sickle cell anaemia (SS) patients with low (<9%) HbF levels (LFSS) are characterized by an increased number of circulating BFU-E in active DNA synthesis, and release of burst promoting activity (BPA) by unstimulated low density (LD) adherent cells. In contrast, circulating BFU-E from SS patients with high (>9%) HbF levels (HFSS) are normal in number, largely in resting phase, and their LD cells do not release BPA-like activity.
We report now that in LFSS patients, adherent cell depletion decreases BFU-E growth in culture and apparent BFU-E cycling. Furthermore, addition of conditioned media (CM) from LD cells of LFSS patients restored cycling BFU-E expression in culture. Neutralization analysis with anti-GM-CSF antibody demonstrated that GM-CSF is, at least, one factor responsible for BPA activity present in this CM. Thus, GM-CSF is constitutively produced by unstimulated monocytes in LFSS patients. In contrast, HFSS patients' adherent cell depletion increases cycling of BFU-E in culture. CM from HFSS patients inhibits BFU-E expression in culture. Hence, LD adherent cells from HFSS patients may release a yet unknown inhibitor factor(s). In addition, we report a distinct response pattern in SS patients' BFU-E to growth factor (GM-CSF, IL-3): (a) LFSS patients have a BFU-E population, equally responsive to GM-CSF and IL-3; (b) HFSS patients, have a subset of BFU-E exclusively dependent on IL-3 (20–40% of the circulating BFU-E). This pattern is very similar to that of normal BFU-E.
In conclusion, BFU-E from LFSS patients represent an actively proliferating population, equally responsive to GM-CSF and IL-3, controlled by constitutively produced GM-CSF, suggesting a unique BFU-E behaviour in SS patients with low HbF levels and high haemopoietic stress. The heterogeneous regulation of BFU-E in SS disease seems to be the epiphenomenon of HbF levels, and not vice versa.     

三氧化二砷对5种人类白血病细胞体外效应的比较研究

目的了解三氧化二砷(AS2O3)在体外对K562、BV173、 HL60、U937等白血病细胞生长增殖、凋亡、细胞周期的影响,并与NB4细胞进行比较.方法体外培养上述细胞株,在不同浓度AS2O3作用不同时间后以锥虫蓝拒染计数法计数细胞,绘制生长曲线;通过PI和Annexin V双染色流式细胞仪检测细胞凋亡率;PI染色、流式细胞仪检测药物作用前后白血病细胞的周期分布改变.结果AS2O3明显抑制实验所用NB4、K562、BV173、U937、HL60等 5种白血病细胞的生长增殖,且其效应在2～10 μmol/L的浓度范围内均呈剂量依赖性和时间依赖性;5 μmol/L 浓度AS2O3处理48 h后细胞凋亡率分别为K562(14.6±2.5)%、BV173(19.4±3.1)%、U937(13.8±3.6)%、HL60(18.2±4.0)%,与NB4细胞(41.8±2.6)%比较,差异有统计学意义(均P＜0.01). 以5 μmol/L 浓度的AS2O3经24 h处理,NB4细胞的周期改变以亚G1峰增加为主(5.2%∶36.2%);BV173细胞以G0/G1期细胞比例的增加为主(55.2%∶71.7%),K562细胞及U937细胞以G2/M期细胞比率增加最为明显(分别为22.6%∶45.8%,15.7%∶48.6%).结论在本实验浓度范围内AS2O3对5种白血病细胞株均具有明显生长抑制作用,对不同白血病细胞抑制生长的机制不同,NB4细胞以诱导凋亡为主,K562、BV173、U937白血病细胞以诱导周期停滞为主.提示周期停滞而凋亡不足是AS2O3对NB4以外白血病细胞体外效应的普遍特点;探讨周期停滞及凋亡耐受的机制对提高这些白血病细胞的AS2O3敏感性具有重要意义.     

Synergy between transforming growth factor-beta and tumor necrosis factor-alpha in the induction of monocytic differentiation of human leukemic cell lines

We examined the effect of transforming growth factor-beta (TGF-beta) alone and in combinations with other factors on the growth and differentiation of the human promyelocytic cell line HL60 and the human monoblastic cell line U937. Treatment with TGF-beta alone did not significantly affect growth or differentiation of HL60 cells, while it significantly inhibited proliferation and induced monocytic differentiation of a small percentage of U937 cells. Combinations of TGF-beta and tumor necrosis factor-alpha (TNF-alpha) acted in synergy to inhibit cell proliferation and to induce monocytic differentiation of both HL60 and U937 cells. In contrast, no synergy was observed when HL60 cells were treated with TGF-beta in various combinations with interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and retinoic acid. Examination of TNF-alpha receptor expression on HL60 and U937 cells showed that these cell lines expressed comparable levels of high-affinity TNF-alpha binding sites. Treatment of HL60 and U937 cells with TGF-beta did not induce significant changes in TNF-alpha receptor expression in either cell line. In contrast, HL60 cells expressed much lower levels of TGF-beta receptors than did U937 cells. Treatment of both HL60 and U937 cells with TNF-alpha induced a dose-dependent increase in expression of TGF-beta receptors, suggesting that the synergy between TNF-alpha and TGF-beta may result, at least in part, from upregulation of TGF-beta receptor expression by TNF-alpha.     

Ability of flt3 ligand to stimulate the in vitro growth of primitive murine hematopoietic progenitors is potently and directly inhibited by transforming growth factor-beta and tumor necrosis factor-alpha

The recently cloned flt3 ligand (FL) stimulates the growth of primitive hematopoietic progenitor cells through synergistic interactions with multiple other cytokines. The present study is the first demonstrating cytokines capable of inhibiting FL-stimulated hematopoietic cell growth. Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta l) potently inhibited the clonal growth of murine Lin-Sca-l+ bone marrow progenitors stimulated by FL alone or in combination with granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), interleukin (IL)-3, IL-6, IL-11, or IL-12. TGF-beta 1 inhibited more than 96% of the myeloid colony formation in response to these cytokine combinations, whereas TNF-alpha reduced the number of colonies by 58% to 96% depending on the cytokine by which FL was combined. In addition, both TNF-alpha and TGF-beta 1 inhibited more than 90% of B220+ cell production from B220- bone marrow cells stimulated by FL + IL-7. The effects of TNF-alpha and TGF-beta 1 appeared to be due to a direct effect and on the early progenitors because the inhibition was observed at the single cell level, and because delayed addition of the two inhibitors for only 48 hours dramatically reduced their inhibitory effects. A neutralizing anti-TGF- beta antibody showed the presence of endogenous TGF-beta in the cultures and potently enhanced the ability of FL to stimulate progenitor cell growth in the absence of other cytokines. Agonistic antibodies specifically activating the p75 TNF receptors were more efficient than wild type murine TNF-alpha in signaling growth inhibition of Lin-Sca-l+ progenitor cells, whereas the p55 agonist had less effect than murine TNF-alpha. Finally, TGF-beta increased the number of FL + IL-11-stimulated Lin-Sca-1+ cells in the G1 phase of the cell cycle with 76%, whereas TNF-alpha only had a marginal effect on cell cycle distribution. Thus, TGF-beta, TNF-alpha, and p75 TNF receptor agonists are potent direct inhibitors of FL-stimulated progenitor cell growth in vitro.     

In vivo hematopoietic effects of tumor necrosis factor-alpha in normal and erythroleukemic mice: characterization and therapeutic applications

The effects of recombinant, macrophage-derived, murine tumor necrosis factor-alpha (TNF-alpha) on hematopoiesis in vivo has been examined in normal mice and in Friend virus (FV)-induced erythroleukemic mice. Intravenous (IV) administration of a single dose of recombinant murine TNF-alpha (10(5) U per mouse) significantly suppressed normal and leukemic late-stage erythropoiesis as measured by numbers of mature erythroid colony forming cells (CFU-E) in the bone marrow and spleen and by peripheral blood reticulocyte counts. In normal animals, the immature erythroid (BFU-E), macrophage (CFU-M), and granulocyte- macrophage (CFU-GM) compartments were significantly stimulated by TNF- alpha in both the bone marrow and the spleen. In the bone marrow of leukemic mice, the BFU-E, CFU-GM, and CFU-M progenitor cell compartments were also stimulated by treatment with the monokine. In the spleens of leukemic mice (the primary site of FV leukemia cell accumulation), relative numbers of BFU-E and CFU-GM were increased by TNF-alpha, while those of CFU-M were suppressed. TNF-alpha caused a rapid decrease in the markedly elevated spleen weights of progressively leukemic mice, and in multiple doses it caused complete clinical disease regression in a significant percentage of leukemic animals. The combination of TNF-alpha with interferon-gamma (IFN-gamma) increased the incidence of leukemia regression, compared with TNF-alpha alone. These results show that TNF-alpha exerts a suppressive influence on late-stage erythropoiesis in vivo and suggest that this effect might be exploited in the treatment of acute erythroleukemia, erythroid hyperplasias, and related diseases.     

Antiinflammatory effects of tumor necrosis factor on hematopoietic cells in a murine model of erosive arthritis

Objective

To investigate the mechanisms leading to the influx of inflammatory hematopoietic cells into the synovial membrane and the role of tumor necrosis factor receptor I (TNFRI) and TNFRII in this process in an animal model of rheumatoid arthritis (RA).

Methods

We performed bone marrow transplantations in human TNF–transgenic mice using hematopoietic cells from wild‐type, TNFRI^−/−, TNFRII^−/−, and TNFRI/II^−/− mice as donors and assessed the severity of arthritis histologically. Generation of osteoclasts from the different genotypes was analyzed in vitro and in vivo. Apoptosis was analyzed using annexin V staining as well as TUNEL assays.

Results

Despite lacking responsiveness to TNF in their hematopoietic compartment, mice not only developed full‐blown erosive arthritis but even showed increased joint destruction when compared with mice with a TNF‐responsive hematopoietic compartment. We demonstrated different roles of the 2 different TNFRs in the regulation of these processes. The absence of TNFRI on hematopoietic cells did not affect joint inflammation but markedly attenuated erosive bone destruction via reduced synovial accumulation of osteoclast precursors. In contrast, the absence of TNFRII on hematopoietic cells increased joint inflammation as well as erosive bone destruction via the regulation of osteoclast precursor apoptosis.

Conclusion

Our findings indicate that selective blockade of TNFRI, leaving the antiinflammatory effects of TNFRII unaltered instead of unselectively blocking TNF, might be advantageous in patients with RA.

     

Effects of erythroid differentiation factor on inhibition of erythroid colony-formation (CFU-E and BFU-E) by the sera and on BPA secretion by peripheral mononuclear cells in patients with chronic renal failure]

The effect of erythroid differentiation factor (EDF) on the colony formation of erythroid cells (CFU-E and BFU-E) inhibited by the sera from the patients with chronic renal failure (CRF) was evaluated by methylcellulose assay. EDF restored the inhibited formation of CFU-E colony irrespective of the absence or presence of the accessory cells. In addition, EDF was considered to recover BFU-E colony formation through the burst promoting activity (BPA) secreted by the adherent cells. A suicide experiment using 3H-thymidine revealed that EDF acted to induce BFU-E from resting phase to S phase of the cell cycle. Moreover, the number of circulating BFU-E and the secretion of BPA by the mononuclear cells, both of which were significantly low in CRF patients, were also moderately increased by the addition of EDF. These data suggest that EDF could be utilized as a therapeutic agent for pathogenetic factors except hypoproduction of erythropoietin on the anemia of the patients with CRF.     

TGFβ、TNFα及中性脂质对L-02细胞生物活性的影响

本研究探讨转化生长因子β1（TGFβ）、肿瘤坏死因子α（TNF α）和（或）中性脂质对体外培养肝细胞生物学活性的影响。 1.材料与方法:（1）材料:正常成人肝细胞株（L—02）由中科院上海细胞生物研究所提供。甘油三酯（TG）、极低密度脂蛋白（VLDL）和氧化修饰极低密度脂蛋白（OX—VLDL）购自南京医科大学。人     

Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells

The effects of recombinant human tumor necrosis factor (rH-TNF) on the colony growth of human leukemia progenitor cells (L-CFU), granulocyte- macrophage progenitor cells (CFU-GM), and erythroid progenitor cells (BFU-E) were studied. L-CFU was assayed with leukemia cells obtained from patients with acute myelogenous leukemia. CFU-GM and BFU-E were assayed with bone marrow cells obtained from hematologically normal donors and patients with acute leukemia or non-Hodgkin's lymphoma in complete remission. A dose-dependent growth inhibition of L-CFU as well as CFU-GM and BFU-E was observed by rH-TNF at concentrations of 1 to 100 U/mL. The inhibitory effect on L-CFU was significantly greater than that on CFU-GM. No correlation was observed between the inhibitory effect on L-CFU and the number of colonies formed in the cultures without rH-TNF. Preincubation of the progenitor cells in culture medium containing 20% fetal calf serum with up to 1,000 U/mL of rH-TNF for 24 hours did not result in the inhibition of colony growth of L-CFU or CFU- GM. The inhibitory effect of rH-TNF was neutralized by an anti-rH-TNF murine monoclonal antibody.