Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.     

Differential roles for NF-kappa B in endotoxin and oxygen induction of interleukin-8 in the macrophage

The alveolar macrophage is an important source of interleukin (IL)-8 during pulmonary injury. The IL-8 gene promoter sequence contains nuclear factor (NF)-kappa B, NF-IL6, and activator protein (AP)-1 binding sequences. These sites may have differing regulatory roles in hyperoxia-exposed macrophages than in those stimulated by bacterial lipopolysaccharide (LPS). U-937 and THP-1 macrophage-like cells were exposed to air-5% CO2 or 95% O2-5% CO2, with or without 1.0 microg/ml of LPS, and transfected with an IL-8 promoter-reporter containing NF-kappa B, NF-IL6, or AP-1 mutations. Hyperoxia and LPS caused additive increases in IL-8 production by U-937 cells, whereas THP-1 cells responded only to LPS. An NF-kappa B mutation ablated baseline and O2- and LPS-stimulated reporter activity in both cell lines, whereas NF-IL6 mutations had little effect. An AP-1 mutation had an intermediate effect. LPS, but not hyperoxia, stimulated nuclear translocation of NF-kappa B in both cell lines. Pharmacological blockade of NF-kappa B nuclear translocation ablated LPS-, but not hyperoxia-, stimulated IL-8 production. Although an intact promoter NF-kappa B site is crucial to macrophage IL-8 production, only LPS-stimulated production appears to require additional nuclear translocation of NF-kappa B.     

Regulation of complement factor H synthesis in U-937 cells by phorbol myristate acetate, lipopolysaccharide, and IL-1

The capacity of the human monocyte cell line U-937 to synthesize complement factor H was examined. The kinetics of secretion of factor H into cell culture supernatant were followed by a sensitive solid phase RIA capable of measuring 0.1 ng of protein. Daily secretion of factor H was low and almost linear and was approximately 3 ng of factor H per 10(6) cells. Factor H synthesis was inhibited by cycloheximide but returned to the levels seen in untreated cultures after removal of the inhibitor. LPS and IL-1 both effected a time- and dose-dependent enhancement of factor H synthesis. Induction of U-937 cells with PMA to differentiate into macrophage-like cells also resulted in increased factor H synthesis. RIA of cell lysates, immunofluorescence microscopy, as well as FACS analysis all revealed that factor H Ag was also associated with the U-937 cell membrane. The population of U-937 cells bearing membrane-associated factor H was decreased from 77 to 43% after stimulation for 48 h with LPS (1 microgram/ml). [35S]Methionine metabolic labeling and SDS-PAGE analysis of factor H immunoprecipitates from unstimulated and stimulated culture supernatants and cell lysates demonstrated a major polypeptide, m.w. 150,000, and a minor component, m.w. 42,000. Western blot analysis of factor H in fresh normal plasma also detected both 150,000 and 42,000 m.w. factor H proteins. This is in agreement with the recent demonstration of a 4.4- and 1.8-kb mRNA for factor H in human liver. These data demonstrate that U-937 cells synthesize factor H that is structurally and antigenically similar to factor H in normal plasma. The exact nature of the membrane-bound factor H and its functions and mechanism of attachment to the cell membrane remain to be elucidated.     

The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o     

Time dependent production of cytokines and eicosanoids by human monocytic leukaemia U937 cells; effects of glucocorticosteroids

In the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cytokines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol-12 myristate acetate (PMA) for 24 h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 microg ml(-1)) and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10(-11), 10(-9) and 10(-7) M) for 96 h. The production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF-alpha) reached its maximum between 6 and 12 h. Interferon-gamma (IFN-gamma), interleukin-10 (IL-10) and leukotriene B4 (LTB4) were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone) completely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells.     

Immunomodulatory role of IL-4 on the secretion of Ig by human B cells

The effect of IL-4 on the production of Ig by human B cells was examined. Highly purified B cells were stimulated with Staphylococcus aureus (SA) and IL-4 alone or in combination with various other cytokines and the supernatants assayed for Ig by isotype-specific ELISA. IL-4 (10 to 100 U/ml) did not support Ig secretion by SA-stimulated blood, spleen, or lymph node B cells, whereas IL-2 supported the production of all isotypes including IgE. Moreover, IL-4 suppressed the production of all isotypes of Ig by B cells stimulated with SA and IL-2 including IgG1, IgG2, and IgE. IL-4-mediated suppression was partially reversed by IFN-gamma or -alpha and low m.w. B cell growth factor. TNF-alpha and IL-6 did not reverse the IL-4-induced suppression of Ig production. The inhibitory action of IL-4 on Ig production appeared to depend on the polyclonal activator used to stimulate the B cells. Thus, Ig secretion by B cells activated by LPS and supported by IL-2 was not inhibited by IL-4. Whereas IL-4 alone supported minimal Ig production by LPS-activated B cells, it augmented production of all Ig isotypes in cultures stimulated with LPS and supported by IL-2. IFN-gamma further enhanced production of Ig in these cultures. When the effect of IL-4 on the responsiveness of B cells preactivated with SA and IL-2 was examined, it was found not to inhibit but rather to promote Ig production modestly. A direct effect of IL-4 on the terminal differentiation of B cells was demonstrated using B lymphoblastoid cell lines. IL-4 was able to enhance the Ig secreted by an IgA-secreting hybridoma, 219 and by SKW6-CL-4, an IL-6-responsive IgM-secreting EBV transformed B cell line. These results indicate that IL-4 exerts a number of immunoregulatory actions on human B cell differentiation. It interferes with the activation of B cells by SA and IL-2, but promotes the differentiation of preactivated B cells, B cell lines, and B cells activated by LPS without apparent isotype specificity.     

Regulation of monocyte/macrophage C2 production and HLA-DR expression by IL-4 (BSF-1) and IFN-gamma

IL-4 was originally described on the basis of its ability to co-stimulate the proliferation of resting B cells treated with anti-IgM. Recently, this cytokine has been shown to have other effects on mast cells, T cells, B cells, and macrophages. We studied the ability of IL-4 to regulate the production of C2 by human monocytes and monocytic cell lines and compared this with stimulation of HLA-DR expression, another recently described activity of IL-4. Responses to IL-4 were compared to IFN-gamma, a cytokine with both activities. IL-4 up-regulated C2 production by human monocytes and this effect was not inhibited by neutralizing anti-IFN-gamma antibody. IL-4 also stimulated C2 production by HL-60 cells that had been pre-treated with vitamin D3 to induce monocytic differentiation. IL-4 did not stimulate C2 production by U937 cells. IFN-gamma, in contrast to IL-4, stimulates C2 production by all three cell types. Although IL-4 increased C2 production by HL-60 cells we could not detect C2 mRNA by Northern blotting. However, co-stimulation of these cells with IL-4 and low concentrations of IFN-gamma resulted in an additive effect on C2 production and a greater increase in C2 mRNA than was seen with IFN-gamma alone. As reported by others, IL-4-stimulated HLA-DR expression by monocytes. In contrast to our findings regarding C2 production, stimulation of HLA-DR expression was inhibited by neutralizing anti-IFN-gamma mAb and IL-4 did not stimulate HLA-DR expression by U937 or HL-60 cells. IFN-gamma stimulated HLA-DR expression by all three cell types. These results identify IL-4 as an additional cytokine able to directly stimulate C2 production by human monocytes and by a monocytic cell line whereas IL-4 stimulation of HLA-DR expression by monocytes appears to be IFN-gamma dependent.     

Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.     

A human macrophage hybridoma producing a cytotoxic factor distinct from TNF,LT, and IL-1

Summary A stable human macrophage hybridoma was established by somatic cell fusion between human peripheral blood monocyte-derived macrophages and an 8-azaguanine resistant clone of a human histiocytic lymphoma cell line U-937 (clone U-937-F9). The hybrid cell line (F9P) exhibited typical macrophage-like morphology and had 30 more chromosomes than U-937-F9 cells. Its macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mo-2 and LEU-M3 antigens on the cell surface, and the demonstration of phagocytic activity. Furthermore, when stimulated with lipopolysaccharide (LPS), this cell line could secrete a considerable amount of a cytotoxic factor (CTF). Distinct from the hybrid cell line, the parental U-937-F9 cells expressed neither Mo-2 nor LEU-M3 antigens on the cell surface, did not show phagocytic activity, and their culture supernatants did not show cytotoxic activity even after LPS stimulation. The activity of CTF in the culture supernatant of the LPS-stimulated hybrid cells could not be neutralized with anti-tumor necrosis factor, anti-interleukin-1, or anti-lymphotoxin antibodies. The CTF had a relative molecular mass of 45–60×10³ daltons as determined by gel filtration on a column of Superose 12, and an isoelectric point of 5.1. The cytotoxic activity was also induced when the hybrid cells were stimulated with the concentrated supernatants of a human T-cell hybridoma containing macrophage activating factor for cytotoxicity or with LP3 tumor cells which were used as target cells.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

Influence of recombinant IL-4, IFN-alpha, and IFN-gamma on the production of human IgE-binding factor (soluble CD23)

This study documents the influence of rIL-4, IFN-gamma, and IFN-alpha on the production of IgE-BF and the expression of lymphocyte receptor for IgE or CD23 Ag (Fc epsilon R II) by human mononuclear cells. IL-4 increases the secretion of IgE-binding factor (BF) by highly purified B lymphocytes, adherent cells, and U937 monoblastic cells. The effect of IL-4 on purified B cells is augmented by costimulating the cells with F(ab')2 anti-IgM. IFN-gamma, IL-2, IL-1-alpha, or IL-1 beta and the low m.w. B cell growth factor have no effect on IgE-BF production by purified B cells even when they are used in combination with anti-IgM. Stimulation of purified T cells with IL-4 or IL-4 plus PMA leads to the production of very small amounts of IgE-BF that might well be derived from the contaminating non-T cells. IFN-gamma increases IgE-BF synthesis by unfractionated PBMC, T cell-depleted PBMC, adherent cells, and U937 cells suggesting that it induces monocytes to release IgE-BF, IFN-gamma suppresses the IL-4-induced Fc epsilon R II expression and IgE-BF production by highly purified B cells but not by PBMC or their T cell-depleted fractions. IFN-alpha inhibits IgE-BF production by IFN-gamma-stimulated PBMC and by IL-4-stimulated cells suggesting that it exerts its effect on B cells and on monocytes. Moreover IFN-alpha suppresses the IL-4-induced expression of Fc epsilon R II on B cells. Both IFN-alpha and IFN-gamma suppress the synthesis of IgE by PBMC in response to IL-4. Taken collectively the results indicate that: 1) IL-4 induces IgE-BF production by both B cells and monocytes, 2) IFN-gamma stimulates IgE-BF synthesis by monocytes but suppresses its production by IL-4-stimulated B cells, and finally 3) IFN-alpha inhibits IgE-BF synthesis in response to either IFN-gamma or IL-4.     

Regulation of CD93 cell surface expression by protein kinase C isoenzymes

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.     

Phenytoin induces interleukin-1 production in vitro

Human adherent mononuclear cells and subcloned cell lines established from the human histiocytic cell line U-937 were cultured with phenytoin (PHT) and/or lipopolysaccharide (LPS) purified from Bacteroides fragilis. After the cultivation period, the cell-free supernatants were tested for interleukin-1 (Il-1) activity. The results showed that PHT induces Il-1 activity and potentiates LPS-induced Il-1 production. In the monocytic cell line U-937, the induced Il-1 production was found to be clonally distributed indicating that the response to PHT may be exerted by a subpopulation of monocytes. The PHT-induced Il-1 activity may be of importance in the development of gingival overgrowth. The induced Il-1 could also contribute to other known side effects such as dermatologic reactions accompanied by transient fever seen in patients medicating the drug.     

IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells

Murine IL-10 (cytokine synthesis inhibitory factor) inhibits cytokine production by Th1 cell clones when they are activated under conditions requiring the presence of APC. By preincubating APC with IL-10, we demonstrate that IL-10 acts principally on APC to inhibit IFN-gamma production by Th1 clones. Moreover, IL-10 is not active when Th1 cells are stimulated with glutaraldehyde-fixed APC, which also indicates that its action involves regulation of APC function. Furthermore, IL-10 inhibits cytokine synthesis by Th1 cells stimulated with the super-antigen Staphylococcus enterotoxin B, which does not appear to require processing. Flow microfluorimetry purified splenic or peritoneal B cells and macrophages, and B cell and macrophage cell lines can present Ag to Th1 clones. However, IL-10 acts only on sorted macrophages and the macrophage cell line to suppress IFN-gamma production by Th1 clones. IL-10 does not show this effect when B cells are used as APC. In contrast, IL-10 does not impair the ability of APC to stimulate cytokine production by Th2 cells. IL-10 does not decrease IFN-gamma-induced I-Ad levels on a macrophage cell line. Inasmuch as IL-10 also inhibits IL-2-induced IFN-gamma production by Th1 cells in an Ag-free system requiring only the presence of accessory cells, these data suggest that IL-10 may inhibit macrophage accessory cell function which is independent of TCR-class II MHC interactions.     

Establishment of a macrophagelike cell line derived from U-937, human histiocytic lymphoma,grown serum-free

Summary A human macrophagelike cell line which grows in serum-free medium was established from a histiocytic lymphoma cell line, U-937. U-937 cells failed to differentiate into macrophagelike cells in serum-free medium plus 12-O-tetradecanoyl phorbol 13-acetate (TPA). Fibronectin and albumin in serum were necessary for differentiation of U-937 ceds into macrophagelike cells in enriched RDF medium supplemented with insulin, transferrin, ethanolamine, selenite, egg yolk lipoprotein (eRDF-ITESL medium). The established cell line exhibited several characteristic properties of macrophage such as nitroblue tetrazolium reduction, phagocytic and α-naphthylbutyrate-esterase activities, and tumor necrosis factor and interleukin 1 production. At present the cells have been continuously maintained in eRDF-ITESL medium through over 150 passages.     

Up-regulation and down-regulation of cell surface and mRNA expression of CD5 antigen by various humoral factors on murine 70Z/3 pre-B cell leukemia cell line: IL-4 down-regulates CD5 antigen expression.

CD5, a pan-T cell antigen, is expressed on a minor subset of normal B lymphocytes and on cells of most B lineage tumors or transformed B cells in both man and animal models. In the present study, the effects of various humoral factors on CD5 expression by cells of a subcloned 70Z/3 murine pre-B leukemia cell line were investigated. Among the humoral factors studied, only LPS up-regulated CD5 expression on 70Z/3 cells (three- to fourfold) in a dose-dependent manner. However, this up-regulatory effect of LPS was not observed when cells were cultured in serum-free medium. NZB-serum factor (NZB-SF), a cytokine we have identified and shown to enhance the maturation and proliferation of immature B cells, synergistically enhanced CD5 expression in the presence of suboptimal doses of LPS. IL-4 down-regulated CD5 expression by 70Z/3 cells induced by LPS or LPS plus NZB-SF in a dose-dependent manner. IL-4 also suppressed spontaneous CD5 expression by 70Z/3 cells. No other cytokine tested showed an inhibitory effect. LPS, IFN-gamma, NZB-SF, and IL-1 enhanced sIg expression on 70Z/3 cells and their action on sIg expression was not inhibited by IL-4. Thus, the down-regulatory action of IL-4 on CD5 expression appeared specific for this antigen. IFN-gamma, which inhibits IL-4 induced CD23 and DR expression on B cells, does not abolish the down-regulatory action of IL-4 on CD5 expression by 70Z/3 cells. Changes in mRNA levels on coding CD5 were also examined following the incubation of 70Z/3 cells (24 hr) in the presence of humoral factors which can influence CD5 Ag expression. The levels of mRNA for CD5 Ag were moderately increased in the presence of LPS and NZB-SF. IL-4 appeared to suppress the actions of NZB-SF and LPS at least in part by reducing the levels of mRNA encoding CD5.     

Characterization of accessory cell costimulation of Th1 cytokine synthesis.

We studied the capacity of macrophage and B cell lines to provide a costimulatory signal that enhances synthesis of IFN-gamma and IL-2 by mouse Th1 clones stimulated with suboptimal doses of immobilized anti-CD3 antibody. The J774 macrophage line and the CH27 B lymphoma line had the greatest costimulatory activity and routinely increased IL-2 production by 10-fold to 100-fold. Other macrophage and B cell lines had less activity and T cell lines were unable to costimulate. The J774 and CH27 lines did not costimulate IL-4 production by a Th2 clone and had only a small effect on IL-2 production by T cell hybridomas. The process of costimulation was fixation-sensitive, contact-dependent and did not involve stable cytokines present in the T cell/accessory cell conditioned media. Neutralizing antibodies for IL-1, IL-6, and TNF failed to inhibit costimulation. Antibodies to the LFA-1/ICAM-1 pair of adhesion molecules also failed to inhibit. Costimulation of IL-2 production by accessory cells was found to have a unidirectional species restriction: mouse accessory cells costimulated mouse and human IL-2-producing T cells, but human U937 cells induced with PMA were effective only for human T cells. The results indicate that accessory cells can significantly regulate Th1 effector function at the level of cytokine production.     

IFN-gamma stimulates IgG2a secretion by murine B cells stimulated with bacterial lipopolysaccharide

rIFN-gamma strikingly enhances the secretion of IgG2a by murine splenic B cells stimulated with bacterial LPS in vitro and concomitantly suppresses the production of IgG3, IgG1, IgG2b, and IgE while sparing IgM secretion. IFN-gamma stimulates highly purified B cell populations to secrete IgG2a, strongly suggesting that it acts directly on B cells. It increases the frequency of precursors of IgG2a-expressing soft agar colonies and enhances the number of IgG2a+ cells in colonies indicating that it both increases the frequency of precursors of IgG2a+ cells and enhances the number of IgG2a+ daughter cells emerging from each precursor. IFN-gamma completes its action within the first 24 to 48 h of a 6-day culture with LPS and its addition cannot be delayed beyond the first 48 h. Preincubation of resting B cells in the presence of IFN-gamma leads to a time dependent increase, up to 42 h, in IgG2a secretion upon subsequent addition of LPS. IFN-gamma can exert this action on resting B cells that have been selected for absence of membrane IgG expression by cell sorting. The promotion of IgG2a secretion appears to be a specific property of IFN-gamma in that IFN-alpha, IFN-beta, IL-1, IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage-CSF, granulocyte-CSF, and CSF-1 fail to enhance IgG2a secretion by LPS-stimulated B cells.