Tumour necrosis factor-alpha and lipopolysaccharide enhance interferon-induced tryptophan degradation and pteridine synthesis in human cells

The capacity of recombinant interferon-alpha, -beta and -gamma, of bacterial lipopolysaccharide and of recombinant tumour necrosis factor-alpha to induce indoleamine 2,3-dioxygenase and synthesis of pteridines was studied in human peripheral blood mononuclear cells, human macrophages and normal dermal fibroblasts. The action of interferon-alpha and -beta on macrophages was supported by lymphocyte factors as indicated by the effect of these mediators in the absence or presence of lymphocytes. Tumour necrosis factor-alpha alone was ineffective in peripheral blood mononuclear cells and macrophages, but it significantly increased the action of all three interferon species on macrophages and fibroblasts. Lipopolysaccharide directly affected macrophages or dermal fibroblasts and enhanced the effect of interferon-gamma. However, in the presence of lymphocytes, the action of lipopolysaccharide was mediated via interferon-gamma.     

Effect of interferons on the activity of human tumor degeneration factor (TDF)

KB cells were cultivated in the well with tumor-degenerating factor (TDF) and the natural and recombinant interferons. TDF alone induced the degenerative changes of the KB cells and formed the cell-free area, but the interferons alone did not induce the changes and did not form the cell-free area. When KB cells were cultivated with TDF and natural interferons (HuIFN-alpha, -beta and -gamma), the cell-free area was enlarged. Particularly, HuIFN-gamma enhanced the TDF activity most strongly. The recombinant interferons (HuIFN-alpha, -beta and -gamma) also augmented in the same ways as the natural interferons.     

Demonstration of synergy between the factor stimulating granulocytes and macrophages colonies and interleukin-I for the stimulation of prostaglandin E2 synthesis by bone marrow cells

Conditioned medium from P388 D1 cell line containing interleukin 1 (IL-1) and granulocyte macrophage colony stimulating factor (GM-CSF) can stimulate prostaglandin E2 (PGE2) production by murine bone marrow cells. In this work, we show that although GM-CSF (either purified from P388 D1 CM or murine recombinant GM-CSF) does not significantly alter bone marrow cell PGE2 production, its presence in P388 D1 CM is however necessary to induce this effect since the presence of anti GM-CSF antiserum completely abrogated the increase in PGE2 production in response to P388 D1 CM. In addition IL-1 tested alone does not not modify PGE2 release by bone marrow cells. However, the simultaneous addition of IL-1 and GM-CSF markedly increases PGE2 production. Thus, the ability of P388 D1 CM to stimulate PGE2 synthesis by bone marrow cells appears to result from a synergistic action between GM-CSF and IL-1.     

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.     

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.     

The synergistic influence of human interferon-gamma and interferon-alpha on suppression of hematopoietic progenitor cells is additive with the enhanced sensitivity of these cells to inhibition by interferons at low oxygen tension in vitro

The influences of human interferons--natural gamma (2 X 10(7) NIH reference U/mg), recombinant gamma (approximately 5 X 10(6) U/mg), natural alpha (1.4 X 10(8) international reference U/mg), and natural beta (10(6) international reference U/mg)--were evaluated alone or in combination for their effects in vitro on colony formation by low density human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in normal incubator (approximately 20%) O2 tension or low (5%) O2 tension. Alone, these interferons demonstrated the same dose response inhibitory curves, as we reported previously, when cells were grown at 20% O2. Recombinant IFN-gamma gave the same dose response curve as natural IFN-gamma. Natural or recombinant interferon synergized with IFN-alpha to suppress colony formation at concentrations that were approximately 2 log units lower than that required by either interferon alone. Equal concentrations of these interferons were not needed for the synergistic effect and were still apparent when one was present at concentrations of 2 log units less than the other. IFN-gamma synergized to a lesser extent with IFN-beta, but IFN-alpha did not synergize with IFN-beta. Cells grown at 5% O2 were more sensitive to inhibition by 2 log units less IFN-gamma or IFN-alpha, and this effect was additive with the synergistic effects of IFN-gamma and IFN-alpha together. These results may have physiological, pathological, and/or clinical relevance.     

Generation of cytotoxic cells from murine bone marrow by human recombinant IL 2

Murine bone marrow (BM) cells were cultured in recombinant IL 2 (rIL 2) and interferon-alpha, -beta, and -gamma, and cytotoxic activity against YAC cells was determined in a 4-hr 51Cr-release assay. rIL 2 at 20 U/ml was the only lymphokine that consistently induced significant cytotoxic activity within 3 days of culture, peaking around 5 to 7 days. The cytotoxic cells generated are heterogeneous, consisting of at least two populations of cells: a) NK-1+, Qa-5+, AsGm-1+ Thy-1+/-, Lyt-2- cells, similar to natural killer (NK) cells, and b) NK-1-, Qa-5+, AsGm-1+ Thy-1+, Lyt-2+ cells, similar to cytotoxic T lymphocytes. The precursor/accessory cells of these BM cytolytic cells maintained in 20 U/ml of rIL 2 were Qa-2+, Qa-5+, Thy-1+/-, AsGM-1+/-, and NK-1+/- but Lyt-2-. They also lysed NK-resistant targets, P815 and BW5147, and the antigenic phenotypes of these cells were similar to those that lysed YAC cells. These studies indicate that IL 2 alone is adequate to generate cytotoxic activity from BM and that these cytotoxic cells were similar to splenic NK cells.     

Enhancement of cell growth by human interferons

The effects of human interferons (HuIFN) on the human osteosarcoma cells were examined. HuIFN-alpha, -beta and -gamma enhanced dose-dependently the cell growth. There was no difference in the degree of the enhancement of the cell growth among HuIFN-alpha, -beta and -gamma. The higher the cell density was, the lower the degree of the enhancement of the cell growth. When HuIFN-gamma was neutralized with anti-HuIFN-gamma, the enhancement of cell growth was not found.     

Interferons and bone. A comparison of the effects of interferon-alpha and interferon-gamma in cultures of human bone-derived cells and an osteosarcoma cell line

Recombinant human interferon-alpha 2C and recombinant human interferon-gamma (5-1000 U/ml) inhibit the proliferation of normal human bone-derived cells and a human osteosarcoma cell line. In the bone-derived cells the inhibitory effect of interferon-gamma was significantly greater than that of interferon-alpha, whereas in the osteosarcoma cell line the inhibitory effects of both interferons were quantitatively similar. Interferon-alpha did not affect the alkaline phosphatase activity of either type of cells. In contrast, interferon-gamma affected the activity of the enzyme in both cell types: in the bone-derived cells the effect of interferon-gamma was stimulatory whereas in the osteosarcoma cells the effect was inhibitory. In both cell types interferon-gamma selectively inhibited the incorporation of radiolabelled proline into type I collagen. In the osteosarcoma cells, the effects of both interferons on collagen synthesis were quantitatively similar. In the bone-derived cells, however, interferon-alpha decreased proline incorporation into collagen and non-collagen proteins to a similar extent and thus did not affect collagen synthesis when expressed as a percentage of total protein synthesis. Two-dimensional polyacrylamide gel electrophoresis of the radiolabelled proteins of the cell layer synthesised by both cell types in the presence of either interferon demonstrated that this treatment enhanced or induced the synthesis of a total of 21 individual proteins (19 in bone cells, 14 in osteosarcoma), ranging in apparent molecular mass over 14-87 kDa. The set of proteins induced was different in all four combinations of cells and interferon. A tentative identification of several of the proteins was possible based upon estimation of molecular mass, preferential induction by interferon-alpha or interferon-gamma and differential induction in normal and transformed bone-derived cells. The results of this study demonstrate that interferons have complex effects upon the proliferative and biosynthetic activities of human bone-derived cells and demonstrate significant differences between the responses of normal cells and transformed bone-derived cell line. Further investigations will be required in order to determine whether or not these differences are unique to the osteosarcoma cell line or are a characteristic of the effects of interferons on bone-derived cells in general.     

Induction of indoleamine dioxygenase by interferon in mice: a study with different recombinant interferons and various cytokines

Since it is important the availability of a specific marker for interferon induction in vivo, we investigated the effect of different recombinant interferons and various cytokines on indoleamine 2,3-dioxygenase activity. Although with different magnitude, recombinant interferon-alpha A/D (Bgl II) hybrid, interferon-gamma and tumor necrosis factor, all increase the activity of this enzyme, whereas interleukin-1, recombinant interferon-alpha A and interferon-alpha D do not induce this activity in mice lung tissue. Dexamethasone is able to inhibit indoleamine 2,3-dioxygenase induction by lipopolysaccharide or by interferon-alpha A/D but it fails to prevent the induction by interferon-gamma.     

Anti-tumor cytotoxic potential and effect on human bone marrow GM-CFU of human LAK cells generated in response to various cytokines.

The effects of various recombinant cytokines i.e. IL-1 alpha, IL-3, IL-4, IL-6, IFN-gamma, TNF-alpha and GM-CSF used either alone or in combination with IL-2, were investigated in this study. First, their capacity to induce killer cells from human PBL was examined by evaluating the degree of killing of human NK-sensitive K562 or NK-resistant Daudi cells. Second the effects of these cytokines, LAK cells (at 1/1, 2/1, 4/1 ratio LAK effectors/bone marrow cell targets) and of the supernatants from washed killer cell cultures, were examined on the colony forming ability of human bone marrow for GM-CFU in vitro. Various degrees of NK activity against K562 was observed in PBL stimulated with the cytokines, whereas LAK activity was found only with IL-2 alone. Culture of PBL with IL-2 + IL-1 alpha or IL-2 + IL-6 or IL-2 + GM-CSF resulted in the highest LAK killing. However, addition of TNF-alpha, or IFN-gamma to IL-2 in cultures resulted in a significant suppression of LAK cell activity. Addition of IL-1 alpha, IL-2, IL-3, and IL-4 to BM cultures had little or no effect on day 14 GM-CFU, whereas addition of IL-6 and GM-CSF resulted in a stimulatory effect. LAK cells induced with IL-2 alone had no significant suppressive effects on GM-CFU.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative effects of various classes of mouse interferons on macrophage activation for tumor cell killing

The effects of mouse interferon-alpha (MuIFN-alpha), -beta (MuIFN-beta), and -gamma (MuIFN-gamma) on macrophage activation for tumor cell killing were determined by using proteose peptone-elicited peritoneal macrophages from C3H/HeN and C3H/HeJ mice under conditions that either included or were free of detectable endotoxin. Alone, under the conditions used, none of the interferons was able to activate macrophages directly for tumor cell killing. However, with a second signal provided to responsive macrophages by contaminating endotoxin, added bacterial lipopolysaccharide (LPS), or heat-killed Listeria monocytogenes (HKLM), all three types of interferon induced cytolytic activity, with MuIFN-gamma approximately 500 to 1000-fold more active than either MuIFN-alpha or -beta. Thus, all three interferons were able to prime macrophages for killing but required a second signal before cytolytic activity could be expressed. When MuIFN-gamma was mixed with either MuIFN-alpha or -beta and placed on macrophages, little or no killing developed. Mixtures of MuIFN-gamma with either MuIFN-alpha or -beta did increase the sensitivity of macrophages to triggering by LPS, however, compared with macrophages treated with MuIFN-gamma alone. The results are collectively important because they i) confirm that significant quantitative differences exist between the various interferons with regard to their capacity to prime macrophages for tumor cell killing; ii) indicate that to be an efficient activator each type of interferon must be combined with a second stimulus, such as LPS or HKLM; iii) show that neither MuIFN-alpha nor -beta can provide an efficient second triggering signal for macrophages that are primed by MuIFN-gamma; and iv) document that mixtures of MuIFN-gamma with either MuIFN-alpha or -beta are most efficient at inducing priming, compared with any one of the interferons used alone.     

The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma

The influences of human tumor necrosis factor (TNF) (LuKII), recombinant human TNF-alpha, natural human interferon-gamma (HuIFN-gamma), recombinant HuIFN-gamma, and natural HuIFN-alpha were evaluated alone or in combination for their effects in vitro on colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in lowered (5%) O2 tension. TNF (LuKII) and recombinant TNF-alpha caused a similar dose-dependent inhibition of colony formation from CFU-GM, BFU-E, and CFU-GEMM. Day 7 CFU-GM colonies were more sensitive than both day 14 CFU-GM colonies and day 7 CFU-GM clusters to inhibition by TNF. BFU-E colonies and CFU-GEMM colonies were least sensitive to inhibition with TNF. The suppressive effects of TNF (LuKII) and recombinant TNF-alpha were inactivated respectively with hetero-anti-human TNF (LuKII) and monoclonal anti-recombinant human TNF-alpha. The hetero-anti-TNF (LuKII) did not inactivate the suppressive effects of TNF-alpha and the monoclonal anti-recombinant TNF-alpha did not inactivate TNF (LuKII). The suppressive effects of TNF did not appear to be mediated via endogenous T lymphocytes and/or monocytes in the bone marrow preparation, and a pulse exposure of marrow cells with TNF for 60 min resulted in maximal or near maximal inhibition when compared with cells left with TNF for the full culture incubation period. A degree of species specificity was noted in that human TNF were more active against human marrow CFU-GM colonies than against mouse marrow CFU-GM colonies. Samples of bone marrow from patients with non-remission myeloid leukemia were set up in the CFU-GM assay and formed the characteristic abnormal growth pattern of large numbers of small sized clusters. These cluster-forming cells were more sensitive to inhibition by TNF than were the CFU-GM colonies and clusters grown from the bone marrow of normal donors. The sensitivity to TNF of colony formation by CFU-GM of patients with acute myelogenous leukemia in partial or complete remission was comparable with that of normal donors. When combinations of TNF and HuIFN were evaluated together, it was noted that TNF (LuKII) or recombinant TNF synergized with natural or recombinant HuIFN-gamma, but not with HuIFN-alpha, to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM from bone marrow of normal donors at concentrations that had no suppressive effects when molecules were used alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombinant interferon-alpha, -beta, and -gamma enhance the proliferative response of human B cells

Recombinant interferons (IFN-alpha, -beta, and -gamma) were examined for their effects on B cell activation. Relatively small IgM+ B cells from human blood samples were isolated by fluorescence-activated cell sorting and were used as target cells. Although the interferons themselves were nonmitogenic, each enhanced the proliferative response induced by a mitogenic anti-mu monoclonal antibody, with IFN-beta usually showing the greatest enhancement and IFN-gamma the least. Pretreatment with the interferons primed resting B cells to undergo enhanced DNA synthesis in response to the anti-mu antibody DA4. Conversely, anti-mu pretreatment, followed by IFN treatment, did not induce B cells to enter the S phase. Time-course analysis revealed that IFN could augment the anti-mu response even when added as late as the final 24 hr of a 3-day culture interval. Combinations of IFN-gamma plus IFN-alpha or -beta were synergistic in the anti-mu response, whereas the IFN-alpha plus IFN-beta combination was not. The data suggest that interferons produced by both lymphocytes (IFN-gamma) and nonlymphoid inflammatory cells (IFN-alpha and -beta) can enhance B cell growth via different mechanisms.     

Dendritic cells issued in vitro from bone marrow produce PGE(2) that contributes to the immunomodulation induced by antigen-presenting cells.

Given that preliminary work has indicated that prostaglandins can play a role in modulating dendritic cell (DC) functions, we addressed the prostaglandin E(2) (PGE(2)) biosynthetic capacity of mouse DC produced in vitro from bone marrow cells. We observed production of significant amounts of PGE(2), which was reduced by at least 80% when cells were incubated in the presence of indomethacin, a COX-1 preferential inhibitor. Indeed, when tested by Western blot analysis with specific COX-1 and COX-2 antibodies, only COX-1 expression could be detected in the bone marrow (BM)-DC. For lipopolysaccharide (LPS)-treated BM-DC, inhibition of PGE(2) production by indomethacin or by NS-398 (a COX-2-selective inhibitor) used alone was less potent. After LPS treatment of BM-DC, COX-1 and COX-2 expression was potent, and inhibition of PGE(2) synthesis needed the presence of both indomethacin and NS-398. We also observed that exogenous PGE(2) diminished the expression of MHC class II molecules by BM-DC and that prostaglandin and indomethacin had antagonistic effects on cell proliferation during the mixed lymphocyte reaction using BM-DC as stimulatory cells. This assessment of PGE(2) suggests that endogenous PGE(2) produced by DC might play a role as an immunomodulating factor during the immune response. This hypothesis is sustained by the fact that IL-12 production by BM-DC is modulated by exogenous PGE(2) as well as endogenous prostaglandin, since either the addition of exogenous PGE(2) or the presence of LPS (which increases endogenous PGE(2) synthesis) decreases IL-12 production, while NS-398 (which decreases LPS-induced PGE(2) synthesis) increases IL-12 synthesis.     

Induction of receptors for tumor necrosis factor-alpha by interferons is not a major mechanism for their synergistic cytotoxic response

We have investigated the effects of various interferons on the receptors for recombinant tumor necrosis factor-alpha (rTNF-alpha) and also their effects on rTNF-alpha-mediated cytotoxicity on human cervical carcinoma cell line ME-180. Preincubation of cells with interferon (IFN)-gamma causes a concentration- and time-dependent increase in rTNF-alpha receptor number without any change in the affinity constant of the receptors. The increase in receptor number is caused only by IFN-gamma and not by IFN-alpha or IFN-beta. Approximately 4-6 h of preincubation with IFN-gamma are required for maximum increase in rTNF-alpha binding to the cells, and this increase can be abolished by inhibitors of protein synthesis, suggesting de novo synthesis of rTNF-alpha receptors. The half-life of both uninduced and induced receptors of rTNF-alpha is approximately 2 h, indicating a rapid turnover. The binding of rTNF-alpha to the cells can also be eliminated by pretreatment of cells with trypsin. Following the removal of trypsin, binding of rTNF-alpha gradually increases, and this requires the synthesis of new proteins. The cytotoxic effect of rTNF-alpha on ME-180 cells is potentiated severalfold by the addition of either IFN-alpha, -beta, or -gamma. However, at similar concentrations, relatively higher potentiation of rTNF-alpha cytotoxicity is observed with IFN-gamma as compared to IFN-alpha and IFN-beta. The pre-exposure of cells to IFNs is as effective as co-exposure in enhancing cytotoxic effects of TNF-alpha. The induction of TNF-alpha receptors by IFNs is observed in different cell types regardless of their sensitivity to TNF-alpha, suggesting that increase in receptor number alone is not sufficient for the enhanced cytotoxic response. Because the enhancement of cytotoxic effects of TNF-alpha is observed by all IFNs but receptor induction in ME-180 cells occurs only with INF-gamma and because metabolic inhibitors which down-regulate TNF-alpha receptors also enhance cytotoxic response, we suggest that the induction of TNF-alpha receptor by IFNs is not a major mechanism of synergism between these cytokines.     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

Chondrocyte activation by interleukin-1: analysis of the synergistic properties of fibroblast growth factor and phorbol myristate acetate

Following activation, monolayers of lapine articular chondrocytes secreted into their culture media large amounts of prostaglandin E2 (PGE2) and the neutral metalloproteinases collagenase and gelatinase. Partially purified preparations of synovial "chondrocyte activating factors" (CAF), which contain interleukin-1 (IL-1), generally proved stronger activators of chondrocytes than recombinant, human, IL-1 alpha (rHIL-1 alpha) or IL-1 beta (rHIL-1 beta). The presence of synergistic cytokines within the synovial material provides one possible explanation of this discrepancy. As first reported by K. Phadke (1987, Biochem. Biophys. Res. Commun. 142, 448-453) fibroblast growth factor (FGF) synergized with rHIL-1 in promoting the synthesis of neutral metalloproteinases. In our hands FGF alone did not induce neutral metalloproteinases and increased PGE2 synthesis only modestly. However, at doses from 1 ng/ml to 1 microgram/ml, FGF progressively enhanced the synthesis of PGE2, collagenase, and gelatinase by chondrocytes responding to rHIL-1. Acidic and basic FGF synergized equally well with both rHIL-1 alpha and rHIL-1 beta. Phorbol myristate acetate (PMA), but not the Ca2+-ionophore A23187, could substitute for FGF as a synergist. PMA alone was a poor inducer of collagenase or gelatinase but, unlike FGF, it greatly enhanced the synthesis of PGE2 by chondrocytes. Dot-blot analyses with a cDNA probe to collagenase mRNA confirmed that partially purified synovial CAF induced collagenase mRNA more effectively than rHIL-1, with rHIL-1 alpha being superior to rHIL-1 beta in this regard. The synergistic effects of both FGF and PMA upon IL-1-mediated collagenase induction were associated with increased abundance of collagenase mRNA.     

Murine mast cell colony formation supported by IL-3, IL-4, and recombinant rat stem cell factor, ligand for c-kit.

Recently, a novel cytokine designated stem cell factor (SCF) was isolated from medium conditioned by buffalo rat liver cells and proved to be the ligand for c-kit. We have examined the effects of recombinant rat SCF alone and in various combinations with interleukin-3 and interleukin-4 on murine mast cell colony formation in methylcellulose culture. As a source of connective tissue-type mast cells (CTMC), we used peritoneal mast cells. No individual factor supported colony formation by purified peritoneal mast cells. When cells were grown in combinations of two factors, significant mast cell colony growth was seen. When cells were grown in the presence of three factors, not only the number of colonies was increased but also the colonies were larger. Mast cells in these colonies contained safranin- and berberine sulfate-positive cells, but the proportions of positive and negative cells varied depending on the factor combinations. We then examined the effects of these factors on proliferation of bone marrow-derived mast cells (BMMC) by replating pooled mast cell colonies. As a single factor, only interleukin-3 supported mast cell colony formation. Combinations of two of the three factors supported mast cell colony formation. However, the most impressive synergism was seen again with the combination of the three factors. Not only was the number of colonies increased, but there was a significant increase in size. These results indicate that SCF is an important factor for the proliferation of both CTMC and BMMC.     

Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro.

We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony-stimulating activity under fetal bovine serum (FBS)-deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) to stimulate colony growth. Colony morphology was determined by the late-acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL-3 and Epo resulted in the largest number of mixed-cell colonies--a larger number than observed with IL-3 and Epo alone even in FBS-supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed-cell colony-forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.