Epstein-Barr virus vector-mediated gene transfer into human B cells: potential for antitumor vaccination.

The efficient gene transfer of immunostimulatory cytokines into autologous tumor cells or the transfer of tumor-associated antigens into professional antigen-presenting cells is a prerequisite for many immunotherapeutic approaches. In particular with B cells, the efficiency of gene uptake is one of the limiting factors in cell-based vaccine strategies, since normal and malignant human B cells are commonly refractory to transducing gene vectors. Due to its natural tropism for human B cells, Epstein-Barr virus (EBV), a human herpes virus, might be an option, which we wanted to explore. EBV efficiently infects human B cells and establishes a latent infection, while the viral genome is maintained extrachromosomally. Although these characteristics are attractive, EBV is an oncogenic virus. Here, we present a novel EBV-derived vector, which lacks three EBV genes including two viral oncogenes and an essential lytic gene, and encodes granulocyte-macrophage colony-stimulating factor (GM-CSF) as a cytokine of therapeutic interest. We could show that EBV vectors efficiently transduce different B-cell lines, primary resting B cells, and tumor cells of B-cell lineage. Vector-derived GM-CSF was expressed in sufficient amounts to support the maturation of dendritic cells and their presentation of model antigens to cognate T-cell clones in autologous settings and an allogeneic, HLA-matched assay. We conclude that the EBV vector system might offer an option for ex vivo manipulation of B cells and gene therapy of B-cell lymphomas.

Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing.

Bispecific Abs (bsAb) are promising immunological tools for the elimination of tumor cells in minimal residual disease situations. In principle, they target an Ag on tumor cells and recruit one class of effector cell. Because immune reactions in vivo are more complex and are mediated by different classes of effector cell, we argue that conventional bsAb might not yield optimal immune responses at the tumor site. We therefore constructed a bsAb that combines the two potent effector subclasses mouse IgG2a and rat IgG2b. This bispecific molecule not only recruits T cells via its one binding arm, but simultaneously activates FcgammaR+ accessory cells via its Fc region. We demonstrate here that the activation of both T lymphocytes and accessory cells leads to production of immunomodulating cytokines like IL-1beta, IL-2, IL-6, IL-12, and DC-CK1. Thus this new class of bsAb elicits excellent antitumor activity in vitro even without the addition of exogenous IL-2, and therefore represents a totally self-supporting system.

G-CSF, GM-CSF and IL-6 levels in cord blood: diminished increase of G-CSF and IL-6 in preterms with perinatal infection compared to term neonates.

AIM: The objectives of this study were 1) to clarify the physiologic regulation of cytokines such as IL-6, G-CSF and GM-CSF in preterm and term neonates and 2) to evaluate the influence of perinatal stress and infection on endogenous cytokine levels. METHOD: We examined cord blood levels of G-CSF, GM-CSF and IL-6 using a bioassay in 43 term and 44 preterm neonates. RESULTS: Compared to normal neonates (G-CSF: mean (m) = 97.6 +/- 16.3 pg/ml; IL-6: m = 20.2 +/- 4.6 pg/ml), we found elevated G-CSF levels in newborns with perinatal stress (m = 247.1 +/- 72.1 pg/ml; p = 0.003) and increased levels for G-CSF (m = 8980.9 +/- 4388 pg/ml; p = 0.0003) and IL-6 (m = 705 +/- 322.3 pg/ml; p = 0.025) in neonates with infection. Term newborns with infection had higher G-CSF levels than preterms (m = 15575 +/- 9374 pg/ml versus m = 5384.1 +/- 4470.9 pg/ml; p = 0.024). G-CSF levels of newborns with infection were correlated with birth weight (r = 0.50; p = 0.024) but not with gestational age (r = 0.40; p = 0.057). GM-CSF was only detectable in cord blood in 4 cases of normal healthy neonates. CONCLUSIONS: The response of G-CSF levels in preterms to infection is diminished. Body cell mass is more important than gestational age to provide high G-CSF levels during states of infection.

Differential modulation of IL-1-induced endothelial adhesion molecules and transendothelial migration of granulocytes by G-CSF.

Granulocyte colony stimulating factor (G-CSF) is widely used for mobilization of haemopoietic stem cells into the peripheral blood. However, little is known about the mechanisms involved in mobilization and the immune modulatory effects of this growth factor. In this report we show that G-CSF down-regulated intercellular adhesion molecule 1 (ICAM-1) induced by Interleukin-1 (IL-1) on human endothelial cells. Interestingly, the G-CSF-mediated down-modulation of IL-1-induced ICAM-1 appeared to be biphasic. In pharmacological concentrations (> 300 ng/ml), and in dose ranges of plasma G-CSF levels above that of nonfebrile healthy individuals (30 pg/ml), a significant decrease in surface ICAM-1 could be observed. This could be explained, at least in part, by an increased autocrine G-CSF production by endothelial cells in response to IL-1 and exogenous G-CSF. In contrast to ICAM-1, IL-1-triggered VCAM-1 expression was superinduced by G-CSF with the optimal concentration of 30 pg/ml. To evaluate the functional significance of these findings, 51Cr adhesion assays with peripheral blood mononuclear cells (PBMC) or granulocytes known to lack the VCAM-1 counter-receptor very late antigen 4 (VLA-4) and IL-1-stimulated endothelial cells, in the presence or absence of G-CSF, were performed. G-CSF could not inhibit the IL-1-induced adhesion of PBMC to endothelial cells, which may be due to the differential adhesion molecule modulation. In contrast, granulocyte adhesion induced by IL-1 could effectively be blocked by co-incubation with G-CSF. Finally, G-CSF also inhibited transendothelial migration of granulocytes through IL-1-activated endothelial cells in a concentration-dependent manner.

Antibodies to the beta 1-integrin chain, CD44, or ICAM-3 stimulate adhesion of blast colony-forming cells and may inhibit their growth.

Early hematopoietic progenitor cells adhere to bone marrow stromal cells (BMSCs) mainly through VLA-4/VCAM-1 interactions. However, many adhesion molecules are expressed by these cell types. Cell adhesion molecules not only mediate adhesion; some are also capable of triggering cellular signaling events. These signals can be induced by several anti-adhesion molecule antibodies. In this study, we investigated the effects of several of such stimulatory antibodies against alpha L (CD11a), alpha 4 (CD49d), and beta 1 (CD29) integrin chains, ICAM-3 (CD50), CD34, CD44, and CD45. All antibodies reacted strongly with CD34-positive bone marrow (BM) cells, but only those against beta 1 integrin (TS2/16, Lia1/2) or CD44 (NKI-P2) reacted with BMSCs. To test the ability of these antibodies to stimulate adhesive interactions, we analyzed their effect on stroma-adherent blast colony-forming cells (Bl-CFCs). We found that TS2/16 (anti-beta 1 integrin), NKI-P2 (anti-CD44), and 152-2D11 (anti-ICAM-3) enhanced adhesion of BM mononuclear cells to stroma (TS2/16:3.4-fold, NKI-P2: 3.8-fold, 152-2D11: 2.6-fold) when compared with isotype-control-treated cells. The increase in stroma-adherent cells was accompanied by an increase in Day 5-7 blast colonies of 3.8-, 2.6-, and 1.9-fold, respectively. One antibody against CD29:Lia1/2 strongly inhibited the formation of blast colonies, an effect that was at least partially caused by its growth-inhibitory activity. Of the other antibodies tested, none displayed growth-modulatory activity. We have found previously that Bl-CFCs depend strongly on VLA-4 and VCAM-1. However, in TS2/16- or 152-2D11-treated cultures, we observed not only these, but also VLA-5-dependent adhesive interactions. In contrast, VLA-5 did not appear to be involved in NKI-P2-treated cultures. Our data indicate that interactions mediated by beta 1-integrins are involved in the growth of Bl-CFCs. Furthermore, interactions mediated by beta 1-integrins, CD44, and ICAM-3 differentially modulate VLA-4- and VLA-5-dependent progenitor/BMSC interactions.

Regulated plasma levels of colony-stimulating factors, interleukin-6 and interleukin-10 in patients with acute leukaemia and non-hodgkin's lymphoma undergoing cytoreductive chemotherapy.

Endogenous plasma levels of granulocyte colony stimulating factor (G- CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF),IL-6 and IL-10 were measured in a total of 70 patients undergoing cytoreductive chemotherapy for treatment of acute leukaemia or non-Hodgkin's lymphomas. the diagnoses were acute myeloid leukaemia (AML; n = 30), acute lymphoblastic leukaemia (ALL;n=6), non-Hodgkin's lymphomas (NHL; n=11) and other malignant haematological disorders including myelodysplastic syndromes (n=23). After chemotherapy, plasma G-CSF was elevated (mean 5.6 ng/ml; range 1.2-10 ng/ml), and was inversely correlated with white blood cell counts (WBC) (r=-0.7, p<0.001). Occurrence of fever (T>38.0 degrees C) during severe myelosuppression (WBC<1x10(9)/1) was associated with an additional increase of G-CSF levels (P<0. (P<0.001). Plasma IL-6 correlated significantly with fever (range <1 to 1100 pg/ml, mean 130 pg/ml; r=0.5, P<0.001) but revealed only a weak association with WBC or platelet counts. In patients treated with recombinant G-CSF (n = 9), an association between IL-6 and fever was still observed after chemotherapy. During the nonfebrile status (total n = 242; AML n = 124), IL-6 levels remained <9 pg/ml in 90% of cases, whereas G-CSF increased with leucopenia (r = -0.72;P<0.001). In contrast, endogenous GM-CSF remained normal and IL-10 showed only a slight increase (21% of samples; maximum 22 pg/ml) in severe leucopenia. In particular, IL-10 levels did not correlate with G-CSF or IL-6 levels. We conclude that systemic release of G-CSF and IL-6 is obviously nit abrogated by cytoreductive chemotherapy in acute leukaemia and NHL may add to the therapeutic efficacy of recombinant cytokines. Also, plasma levels of G-, GM-CSF or IL-6 appear to be regulated by separate mechanisms.

Constitutive and modulated cytokine expression in two permanent human bone marrow stromal cell lines.

We present a detailed analysis of cytokine expression patterns of the two permanent human bone marrow stromal cell lines, L87/4 and L88/5. These cell lines, previously established in our laboratory, are highly radiotolerant without cell detachment and support long-term cultures of CD(34+)-enriched human cord blood cells. RT-PCR analysis of 22 different cytokines or cytokine receptor mRNAs showed an almost identical expression pattern in the two stromal cell lines compared to primary human Dexter-type stroma. Since stromal feeder lines employed in long-term cultures usually are irradiated and grown in media containing corticosteroids, we analyzed the impact of irradiation and dexamethasone on cytokine production in the two cell lines by RT-PCR, Northern blot analysis, bioassays, and RIAs. By RT-PCR analysis, constitutive mRNA expression of c-kit, G-CSF, GM-CSF, IL-1 beta, IL-6, IL-7, IL-8, IL-11, Kit ligand (KL), LIF, M-CSF, MIP-1 alpha, TGF-beta, and TNF-alpha was demonstrated in both cell lines, with L87/4 a more potent cytokine producer than L88/5. Northern blot data showed an increase in mRNA levels for GM-CSF, IL-1 beta, and LIF by irradiation and IL-1 alpha treatment in both cell lines. IL-1 alpha-induced GM-CSF, IL-1 beta, IL-6, IL-11, and LIF mRNA levels were reduced by the addition of dexamethasone, whereas dexamethasone had no influence on the amounts of IL-1 alpha-induced G-CSF mRNA. L87/4 and, to a lower extent, L88/5 cells showed dexamethasone-dependent increases in KL mRNA, while KL mRNA levels were not stimulated by IL-1 alpha.

VLA-4 and VCAM-1 are the principal adhesion molecules involved in the interaction between blast colony-forming cells and bone marrow stromal cells.

The molecular basis and functional significance of interactions between haemopoietic progenitor cells and the stromal microenvironment is still poorly understood. Here we investigated a broad panel of surface adhesion molecules for their involvement. For this purpose, the colony-forming capacity of stroma-adherent Bl-CEC, BFU-E and GM-CFC was studied. Both mononuclear bone marrow cells (BMC) and bone marrow-derived stromal cells (BMSC) express a wide variety of adhesion molecules. However, only antibodies against beta 1-, alpha 4-integrin (both chains of the very late activation antigen-4 (VLA-4)) and vascular cell adhesion molecule (VCAM-1) inhibited colony formation from stroma-adherent Bl-CFC by 50% or more. Antibodies against a panel of other adhesion molecules, including the alpha 5-integrin chain, were without effect. Subsequent pretreatment experiments revealed that VLA-4 on progenitors interacted with stromal VCAM-1. The inhibitory antibodies did not interfere with the clonogenic capacity of but with adhesion of BFU-E and GM-CFC. Whether the inhibitory antibodies act similarly on progenitors which depend on BMSC for growth and/or differentiation, such as BI-CFC, remains to be determined.

IL-6 and G-CSF levels in amniotic fluid during the second trimester in normal and abnormal pregnancies.

Activity levels of cytokines were measured by stimulation of the cell lines NFS-60, 7TD1, and TF-1. In 39 samples of amniotic fluid, levels of Granulocyte-Stimulating Factor (G-CSF) were 1434 +/- 2063 (mean +/- SD) and of Interleukin (IL-6) 546 +/- 1071 pg/ml; Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) was not detectable. IL-6 was correlated to G-CSF (r = 0.3; p = 0.003). G-CSF (p = 0.0002) and IL-6 (p = 0.006) were influenced by Alpha-Fetoprotein (AFP) and G-CSF by rhesus-incompatibility (p = 0.0004). These findings suggest that cytokines such as IL-6 and G-CSF play some role in physiological and pathological pregnancy.

Simultaneous determination of histamine and serotonin in mast cells by high-performance liquid chromatography.

A sensitive and rapid high-performance liquid chromatographic method for the simultaneous determination of histamine and 5-hydroxytryptamine (serotonin) in supernatants and cellular extracts from mouse bone marrow-derived mast cells was developed. The described method is based on a precolumn derivatization of histamine and serotonin with o-phthaldialdehyde (OPA) and subsequent separation of the amine-OPA adducts with an analytical reversed-phase C18 column combined with fluorometric detection (excitation wavelength 360 nm, emission at 455 nm). The mobile phase was 0.02 M sodiumacetate buffer (pH 8) including 5 mM 1-octanesulfonic acid and 50% methanol. The detection limits for histamine and serotonin were 3.3 pmol and 6.9 pmol, respectively (signal-to-noise ratio 2:1). The variations of peak areas of repeatedly injected low or high amounts of amine standards were 23.3% or 6.9% (for 0.5 ng or 12.5 ng histamine) and 26.4% or 4.0% (for 5.0 ng or 250 ng serotonin) during an experimental period of 4 weeks. The variation of retention times over 2 days was 1.1% for histamine and 1.7% for serotonin. A perfect linear relationship of amine concentrations and peak areas was documented by correlation coefficients of r > 0.999 for both histamine (0.1-125 ng) and serotonin (0.5-250 ng).

Granulocyte colony-stimulating factor inhibits the endogenous leukotriene production in tumour patients.

Granulocyte colony-stimulating factor (G-CSF) is virtually devoid of inflammatory side-effects when given to patients in therapeutic doses. This is in contrast to other haemopoietic cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) which may promote inflammatory reactions by increasing the number and/or activity of monocytes, eosinophils, mast cells and basophils. Inflammatory reactions to GM-CSF and IL-3 appear to be related to an increased formation of leukotrienes, known as potent mediators of allergy and inflammation. Here we report that, in contrast to GM-CSF or IL-3, G-CSF has the potential to inhibit the leukotriene production in vivo. G-CSF may thus act as an anti-inflammatory agent. The differential effects of G-CSF and other haemopoietins on endogenous leukotrienes may be of major clinical significance.

Establishment of two permanent human bone marrow stromal cell lines with long-term post irradiation feeder capacity.

We describe the establishment of two permanent Simian virus 40-transformed human stromal cell lines, designated L87/4 and L88/5, derived from the bone marrow of a hematologically normal male patient. Both cell lines show a fibroblastoid morphology and do not express hematopoietic cell markers. L87/4 but not L88/5 expresses the macrophage marker CD68. The most remarkable feature of these new stromal cell lines is their ability to persist as growth-arrested adherent feeder cells after ionizing-irradiation at doses up to, and exceeding 20 Gy (L87/4). This renders them particularly useful for studying aspects of feeder dependence of hematopoietic cell development in long-term culture. Both cell lines are able to function as feeder cells, supporting the long-term proliferation of CD34+ human cord blood cells as well as the clonogenic growth of the human Burkitt lymphoma B-cell line BL70.

Mitogenicity of anti-Thy-1 monoclonal antibodies attributable to an Fc-dependent mechanism.

We analyzed the mechanism by which certain anti-Thy-1 monoclonal antibodies (mAb) activate T cells directly without additional stimuli. Using a panel of rat anti-Thy-1 antibodies which included more than 30 IgG2c mAb, we found that only the IgG2c isotype was able to induce a strong proliferative response in both resting T cells and a T cell lymphoma, suggesting that this form of T cell activation is isotype restricted and might be a consequence of a unique physico-chemical property of the IgG2c heavy chain. Results from surface distribution studies of Thy-1 molecules, following specific interactions with anti-Thy-1 antibodies of different isotypes, again showed that only IgG2c mAb formed Thy-1 aggregates of high valence on the surface of a T cell lymphoma, and such clustering always evoked a biological response. This led us to propose that IgG2c mAb have the inherent tendency to self-associate, probably through homophilic Fc-Fc contacts, and that this feature renders anti-Thy-1 mAb mitogenic. To prove this, we set up cross-inhibition studies with randomly selected mitogenic (IgG2c) and nonmitogenic (IgG2b) anti-Thy-1 mAb. The results clearly demonstrated that IgG2c antibodies enhance their own binding, analogous to the new form of antibody binding that was recently demonstrated between murine IgG3 mAb and a multivalent antigen. Confirmation of this was also provided by IgG2c-derived F(ab')2 fragments, which were unable to cause proliferation. Furthermore, masking the Fc part of cell-bound IgG2c mAb with a monomeric and thus non-aggregating IgG-binding protein A-derived fragment cancelled their mitogenic ability. Finally, induction of T cell proliferation appeared to be independent of cross-linking via Fc gamma R. The results support a model in which noncovalent intermolecular homophilic contacts of the Fc regions of the IgG2c isotype bring about effective aggregation of Thy-1 molecules, thereby stimulating the mitotic apparatus of the cell.

Characterization of hemopoietic cell populations from human cord blood expressing c-kit.

Human cord blood or bone marrow cells expressing the CD34 surface antigen include a population of pluripotent progenitors. We identified and isolated a subpopulation of cells coexpressing CD34 and c-kit, a transmembrane receptor with tyrosine kinase activity. Novel monoclonal antibodies (16A6, 14A3, 3D6) directed against the extracellular domain of c-kit were used for immunofluorescence labeling and sorting of low-density mononuclear cells (MNCs) from umbilical cord blood and bone marrow. The frequency of c-kit-labeled MNCs from cord blood (mean 5.0% +/- 2.1%, n = 16) was similar to that from adult bone marrow (mean 3.7% +/- 1.3%, n = 4). On average, 1.4% of CD34-positive cells were recorded in cord blood and 2.1% in bone marrow MNCs. Roughly 60% of CD34-positive cells coexpressed c-kit. The ability of CD34+/c-kit+ cells to form multilineage colonies (CFU-GEMM) was assayed after sorting with an antibody that did not show any significant effect on c-kit ligand (RL) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation. For CD34+/c-kit+ cells, we found a 20- to 50-fold enrichment as against total MNCs, and a 2-fold enrichment if compared with the CD34+/c-kit-population. To study expression of c-kit in lymphocytic precursors, monoclonal anti-CD7 or anti-CD10 antibodies were used simultaneously. In contrast to CD34-expressing cells, however, no consistent double-labeled subpopulation of lymphocytic cells was detected. Furthermore, coexpression of CD38 (73% +/- 14%, n = 4) or CD33 (29% +/- 12%, n = 5) on a majority of c-kit-positive cells showed their lineage commitment to erythropoiesis and granulocytopoiesis.

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

Transforming growth factor-beta (TGF-beta) has been described as a potent inhibitor of various cell types, among others of primitive haematopoietic progenitors in vitro, and as a negative autocrine regulator of normal B lymphocyte growth and differentiation. In the present study we investigated TGF-beta gene expression in peripheral blood mononuclear cells (PBMC) and in B cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) and from normal controls. Monocyte depleted B-CLL cells expressed constitutively mRNA for TGF-beta 1 and secreted low amounts of TGF-beta activity into the culture medium. Stimulation of cells by phorbol ester noticeably enhanced mRNA levels as well as protein secretion in most cases. TGF-beta activity was of the same magnitude as in normal controls. We next analysed TGF-beta in highly enriched B lymphocytes from B-CLL (95-100% CD19+), and found that TGF-beta secretion was up to 3 times higher than in the original PBMC population. It is discussed that B-CLL cells might escape from negative regulation by TGF-beta and, on the other hand, inhibit normal haematopoietic cell proliferation and thereby achieve a growth advantage in the haematopoietic tissues.

Lactate production and amino acid incorporation in interleukin 3-dependent, factor-deprived hemopoietic murine cell lines.

Interleukin 3 (IL-3) stimulates the growth of various types of hemopoietic progenitors. In vitro, survival of a series of murine cell lines derived from either neoplastic or nonneoplastic hemopoietic tissue shows a strict IL-3 dependence. In order to test the implication of energy metabolism in this dependence as claimed in several studies, intracellular ATP levels as well as accumulative lactate release were measured in the murine hemopoietic lines FDC-P1, 32Dcl.23, DA-1, DA-3, NFS-60, and NFS-78. ATP levels showed little or no changes within 4-6 h of IL-3 starvation. In the absence of IL-3 the accumulative lactate release ranged from 1.4 to 2.6 mM, and in its presence values between 1.5 and 3.4 mM were recorded within 7 h. Only 32Dcl.23 showed an almost complete suppression of lactate release upon IL-3 withdrawal. The cell cycle times of these cell lines determined by flow cytometry ranged between 9 (DA-3) and 24 h (NFS-78). In the presence of IL-3 there was a significant inverse relationship between cell cycle times and lactate production. It is concluded that neither ATP generation nor the metabolic pathway of lactate production, although the latter correlated with proliferative activity in the studied cell lines, is controlled by IL-3. Furthermore, no control by IL-3 of essential amino acid incorporation into proteins was detected in cell lines 32Dcl.23 and NFS-60.

Macrophage colony-stimulating factor (CSF-1) is expressed by spontaneously outgrown EBV-B cell lines and activated normal B lymphocytes.

Human B lymphocytes activated by mitogens or infected by Epstein Barr virus (EBV) have previously been shown to release colony-stimulating activity (CSA) supporting the growth of normal human bone marrow progenitors. We established five different human EBV-B cell lines spontaneously outgrown from nonmalignant peripheral blood cells and long-term bone marrow cultures. CSA derived from all of these lines induces the growth of murine macrophage colonies, whereas virtually no human bone marrow cell progenitors were stimulated. As observed in the tumor cell line MIA PaCa-2, a 4.3-kilobase (kb) transcript was detected in all cases using a human colony-stimulating factor (CSF)-1 probe. Expression of this transcript can be further stimulated within three hours upon addition of phorbol myristate acetate (PMA). The highly purified native protein exerting macrophage colony-stimulating activity (M-CSA) exhibits a molecular size of approximately 75 to 97 Kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The identity of EBV-B cell derived M-CSA with human urinary CSF-1 was confirmed by a complete neutralization of macrophage CSA by an antihuman urinary CSF-1 antiserum. Normal human B lymphocytes purified from tonsils or from mononuclear blood cells also express CSF-1 upon stimulation with Staphylococcus aureus Cowan I. No CSF-1 expression, however, could be detected in normal resting B lymphocytes or in the Burkitt lymphoma cell line RAJI.

Thiol-sensitive mast cell lines derived from mouse bone marrow respond to a mast cell growth-enhancing activity different from both IL-3 and IL-4.

A series of permanent IL-3-dependent cell lines have been established from normal BALB/c or C3H bone marrow using alpha-thioglycerol-supplemented culture medium and PWM-stimulated spleen cell-conditioned medium as a source of IL-3. The cell lines and derivatives cloned in agar resembled "mucosal type" mast cells with respect to phenotypic and functional properties. In this report we demonstrate that in vitro growth of these mast cell lines was not only dependent on IL-3 and synergistically enhanced by IL-4, but in addition regulated by alpha-thioglycerol which could be replaced by 2-ME or cysteamine. We show that these thiol-sensitive mast cell lines respond to a mast cell growth enhancing activity (MEA) present in spleen cell-conditioned medium and acting in concert with IL-3. Partially purified MEA was not able to stimulate the growth of IL-3-dependent 32Dcl.23 cells, IL-2-dependent CTLL-2 cells or the mouse T cell line F4/4K.6 (L3T4+) adapted to grow in purified IL-4. Moreover, 11B11 hybridoma-derived anti-IL-4 mAb specifically neutralizing mouse Il-4 were unable to abolish the bioactivity of MEA. PWM, CSF-1, GM-CSF, IL-1, IL-2, IL-5, IL-6, IL-7, IFN-gamma, TGF-alpha, TNF-alpha, NGF, or EPO did not substitute for MEA in our standard proliferation assay.

Macrophage colony-stimulating activity is produced by three different EBV-transformed lymphoblastoid cell lines.

The lymphoblastoid cell lines BLY 9.84, Gl, and Pl constitutively release a colony-stimulating activity (CSA) which specifically stimulates murine macrophage progenitor cells in vitro. The biochemical characterization of BLY 9.84-derived CSA exhibits a molecular size of apparently 150-200 kDa even under dissociating conditions with 6 M guanidine hydrochloride or inhibition of glycosylation. Production of this CSA is inhibited by cycloheximide and its activity is destroyed by reduction with dithiothreitol. Replating experiments give evidence for a functional similarity with CSF-1 (macrophage colony-stimulating factor).