Blocking of in vitro and in vivo susceptibility to mouse hepatitis virus.

By pretreatment with concanavalin A (Con A) both in vivo and in vitro genetically susceptible mice and their cultured macrophages have been converted to animals and cells which are phenotypically resistant to mouse hepatitus virus (MHV). Con A at 1.0 mg/mouse decreased the mortality from 100% to less than 40% by inducing a prominent inflammatory response, increasing the number of macrophages in the virus inoculation site, and producing a population of macrophages not uniformly susceptible to the virus. In addition, mediators derived from Con A-treated spleen cells conferred resistance to normally susceptible syngeneic macrophages to 100 TCID50 of MHV.

In vitro macrophage manifestation of cortisone-induced decrease in resistance to mouse hepatitis virus.

Genetically resistant G3H mice routinely yielded macrophages that were resistant when grown in 90% horse serum. These mice also routinely yielded macrophages that were susceptible to the same virus, MHV (PRI), in vitro after the mice had been treated with three intraperitoneal doses, of hydrocortisone. Dexamethasone and prednisolone when similarly administered also increased the susceptibility of C3H macrophages taken from the treated animal, but progesterone and testosterone did not. In addition, spleen cells from mice treated with cortisone made the resistant C3H macrophages 100 times more susceptible in vitro. Increased in vitro susceptibility induced in this way by hydrocortisone was reversed by exposure to supernatant fluid removed from cultures of concanavalin A-treated spleen cells.

Recombinant human granulocyte/macrophage colony-stimulating factor activates intracellular killing of Leishmania donovani by human monocyte-derived macrophages.

Recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) obtained from cloned complementary Mo cell DNA and expressed in COS-1 cells activates cultured peripheral blood monocyte-derived macrophages in vitro to become cytotoxic for intracellular L. donovani. The antileishmanial effect of rGM-CSF, which can be completely neutralized by anti-rGM-CSF antiserum, is maximal after 36 h preincubation with the cultured macrophages, compared with that of rIFN-gamma, which reaches its maximum at 72 h of preincubation. The antileishmanial effect of GM-CSF as well as IFN-gamma is independent of detectable amounts of LPS and is not augmented by the addition of 10 or 50 ng/ml of LPS. Simultaneous administration of suboptimal doses of rGM-CSF and rIFN-gamma to monocyte-derived macrophages results in greater antileishmanial activity by these cells than administration of either lymphokine alone, although no enhancement of antileishmanial activity is observed when optimal doses of these two lymphokines are applied together.

Detection of the lymphokine migration inhibitory factor in normal and disease-affected lung by antibody and by its major binding protein, the interferon antagonist sarcolectin.

Human migration inhibitory factor (MIF) is suggested to play a notable role in regulation of macrophage functions in host defense. A major binding component for the lymphokine in human tissue is the interferon antagonist sarcolectin. This high-affinity interaction gives access to MIF by affinity chromatography on immobilized sarcolectin and may be of significance for in situ activity of MIF. Localization of MIF is one step towards answering this question. Labelled sarcolectin and MIF-specific antibodies can be employed to analyze the expression of the factor. Surgical specimens of 74 patients, who underwent lobe/lung resection or diagnostic biopsy, were fixed with buffered formalin and embedded in paraffin. The material consisted of 36 cases of morphologically normal lung parenchyma of patients, suffering from bronchial carcinoma, of 16 cases with sarcoidosis, of 15 cases with tuberculosis and of 7 cases with idiopathic interstitial pneumonitis. The two types of probe to visualize presence of MIF invariably showed the same level of reactivity, underscoring the potential physiological significance of sarcolectin-MIF interaction. In detail, all cases with pneumonitis, most tuberculosis-affected as well as normal cases and 44% of the cases with sarcoidosis were positive. All positive cases with sarcoidosis and some cases from the other groups revealed accessible binding sites for biotinylated MIF.

Identification and characterization of a non-interferon antileishmanial macrophage activating factor (antileishmanial MAF).

A non-interferon lymphokine elaborated from PHA and Con A-stimulated human T-cell hybridoma, T-CEMA, has been found to activate monocyte-derived macrophages for the intracellular killing of L. donovani (antileishmanial MAF). This T-cell hybridoma derived antileishmanial MAF which has an apparent mw of 65,000 and pI of 5.3-5.6, contains neither antiviral activity nor colony stimulating activity. Furthermore, antileishmanial MAF is not neutralized by anti-MIF, anti-IFN-gamma or anti-GM-CSF antibodies.

Human recombinant migration inhibitory factor activates human macrophages to kill tumor cells.

A recombinant form of human migration inhibitory factor (rMIF) obtained from COS-1 cells transfected with MIF-specific cDNA is able to activate cultured human peripheral blood monocytes and monocyte-derived macrophages, in a dose-dependent manner to become cytotoxic for tumor cells in vitro. The cytotoxicity exhibited by macrophages treated with rMIF is > or = 30% above that of cells incubated with control supernatants or with media and peaks 72 hr after the addition of tumor targets. rMIF also induces macrophages to produce tumor necrosis factor (TNF-alpha) and interleukin-1 beta (IL-1 beta). These results demonstrate that rMIF is able to modulate macrophage functions and plays a role in cell-mediated immune response.

Human recombinant migration inhibitory factor activates human macrophages to kill Leishmania donovani.

A recombinant form of the first lymphokine to be discovered, migration inhibitory factor (rMIF) was obtained from COS-1 cells transfected with a cDNA library from a human T cell hybridoma (6). rMIF has an amino acid sequence unrelated to that of any known protein. To learn more about the biology of MIF, we tested its ability to effect the survival of Leishmania donovani in macrophages. We found that rMIF activates blood monocyte-derived macrophages in vitro to suppress the growth of and kill these intracellular parasites. The anti-leishmanial effect (ranging from 50 to 77% reduction of parasites) is maximal when macrophages have been incubated with rMIF 48 to 72 h before infection and is similar to that seen with macrophages activated by IFN-gamma. Of interest, whereas the activation of human macrophages by IFN-gamma is inhibited by IL-4 and not enhanced by LPS, the activation by rMIF is enhanced by LPS but is not inhibited by IL-4. The data presented here demonstrate that rMIF is a potent activator of macrophages and is likely to be critical in cell-mediated immune host defenses.

Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF.

Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.

Partial purification and characterization of a human lymphokine which induces antileishmanial activity in mouse macrophages.

Human blood mononuclear cells stimulated in vitro with concanavalin A secrete a lymphokine which activates mouse macrophages to kill Leishmania enriettii. Supernatants of cells cultivated for 1 day and for 2 days were analyzed for this activity after G-100 gel filtration and isoelectrofocusing. First day antileishmanial activity was found in a MW range of 23,000 with an isoelectric point of 3.6 to 4.0. Second day antileishmanial activity eluted in a peak in the MW range of 23,000 to 65,000 with an isoelectric point of 4.0 to 4.3. This activity could be completely separated from migration-inhibitory factor (MIF) after isoelectrofocusing in first day and in second day supernatants. Antileishmanial activity could be separated from interferon-gamma in first day, but not in second day supernatants.

Antigen-dependent heterogeneity of human migration inhibitory factor.

Human migration inhibitory factor (MIF) produced by peripheral blood mononuclear cells stimulated with purified protein derivative, tetanus toxoid, streptokinase-streptodornase, or Candida albicans antigen was analyzed by gel filtration and isoelectrofocusing. In all cases, supernatants harvested after a 24-hr exposure of the mononuclear cells to the antigen yielded only one MIF species with an isoelectric point of 5. In contrast, isoelectrofocusing of supernatants obtained from cells exposed to the antigen for an additional 24 hr demonstrated that different antigens induce the elaboration of different MIF species. Streptokinase-streptodornase and tetanus toxoid induced the production of one MIF species with an isoelectric point of 5 (pH 5-MIF). Stimulation of cells with Candida antigen elaborated a MIF species with an isoelectric point of 3 (pH 3-MIF). In contrast, stimulation of cells with purified protein derivative induced the production of both pH 3-MIF and pH 5-MIF.

IL-10 inhibits the synthesis of migration inhibitory factor and migration inhibitory factor-mediated macrophage activation.

IL-10, in addition to being a cytokine synthesis-inhibitory factor, is a cytokine that exerts multiple effects on various cell types. Recombinant human migration inhibitory factor (MIF) inhibits the migration of human monocytes as well as that of guinea pig and murine macrophages. In addition, it has recently been shown to activate human monocyte-derived macrophages to suppress the growth of and/or kill both intracellular parasites and extracellular tumor targets in vitro and to have adjuvant activity in vivo. In this study, we examined the interactions between IL-10 and rMIF. We demonstrate that IL-10 reduces the production of MIF from T cells and abolishes rMIF-mediated migration inhibition of human monocytes. Incubation of IL-10 together with rMIF diminishes rMIF-induced intracellular killing of Leishmania donovani by human monocyte-derived macrophages and inhibits nitric oxide production and nitric oxide synthase activity by murine macrophages.

IL-4 inhibits H2O2 production and antileishmanial capacity of human cultured monocytes mediated by IFN-gamma.

The effect of IL-4 on the IFN-gamma-induced state of activation of cultured human monocytes was investigated with regard to their ability to produce hydrogen peroxide and their antileishmanial capacity towards the intracellular parasite Leishmania donovani. IL-4 was found to inhibit the IFN-gamma-dependent hydrogen peroxide production of monocytes. Treatment of monocytes with IFN-gamma (200 to 600 U/ml) for 48 h increased the hydrogen peroxide production fourfold above background. Coincubation of the monocytes with IL-4 (1 to 1000 U/ml) and IFN-gamma (200 to 600 U/ml) inhibited this increase by 50 to 100%. IL-4 alone did not modulate the hydrogen peroxide production of monocytes. Pretreatment of monocytes with IL-4 for 20 min to 3 h was already effective in preventing the IFN-gamma response. Addition of IL-4 not later than 6 h after the start of incubation with IFN-gamma was necessary for an optimal inhibitory effect. IL-4 also inhibited the IFN-gamma-induced antileishmanial capacity of monocytes: IFN-gamma (1000 U/ml) induced a 54 +/- 10% reduction in the number of parasites. Monocytes treated with combinations of IL-4 (100 to 1000 U/ml) and IFN-gamma (1000 U/ml) were unable to reduce the parasite numbers. IL-4 alone did not alter the uptake of Leishmania donovani nor induce antileishmanial activity. These results demonstrate that IL-4 disables human cultured monocytes to respond to IFN-gamma activation.

An acidic lymphokine distinct from interferon-gamma inhibits the replication of herpes simplex virus in human pulmonary macrophages.

Supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells were fractionated by gel filtration and isoelectric focusing. A fraction with an isoelectric point of 2.2-3.3 containing macrophage migration inhibition factor activity inhibited the replication of herpes simplex virus type 1 in human pulmonary macrophages and U937 cells. This fraction did not inhibit the replication of herpes simplex virus in human fibroblasts. Moreover, the ability of this lymphokine fraction to inhibit viral growth in macrophages was not neutralized by antibody against interferon-gamma. These findings identify a macrophage specific antiviral lymphokine which is distinct biochemically and immunologically from interferon-gamma.

Deficient responses of pulmonary macrophages from healthy smokers to antiviral lymphokines in vitro.

The antiviral function of pulmonary macrophages obtained by broncholavage of healthy smokers and nonsmokers was studied. Compared with nonsmokers' cells, smokers' macrophages produced significantly more virus during in vitro infection with herpes simplex virus type 1 (HSV-1). Exposure of macrophages to either antiviral macrophage-activating factor or interferon-gamma for 20 hr before infection resulted in diminished production of HSV-1 by both types of macrophages. However, in contrast to smokers' cells, exposure of nonsmokers' macrophages to these antiviral lymphokines totally prevented viral replication. This difference could not be attributed to diminished adsorption of virus to smokers' macrophages or to an increased proportion of extracellular to intracellular virus in smokers' cell cultures. The effect of smoking on viral infectivity did not appear to be mediated by secretion of a soluble factor by the macrophage because incubation of nonsmokers' cells with supernatant from smokers' cell cultures did not affect the growth of HSV-1.

Recombinant human migration inhibitory factor has adjuvant activity.

Recombinant human migration inhibitory factor (MIF), isolated through functional expression cloning in COS-1 cells, up-regulates expression of genes encoding HLA-DR and interleukin 1 beta (IL-1 beta) and elaboration of IL-1 beta by human monocyte-derived macrophages. Administration of soluble bovine serum albumin or human immunodeficiency virus 120-kDa glycoprotein (HIV gp120) to mice in the presence of recombinant MIF together with incomplete Freund's adjuvant induced a strong T-cell proliferative response comparable to that of complete Freund's adjuvant. Recombinant MIF also increased antibody production, especially of IgG1 and IgM, in mice. Taken together, these results indicate that recombinant MIF may be useful as an adjuvant in the development of vaccines.

Studies on human migration inhibitory factor: characterization of three molecular species.

Human migration inhibitory factor (MIF), obtained from supernatants of peripheral blood mononuclear cells stimulated with concanavalin A, was analyzed by gel filtration, isoelectrofocusing, and CsCl density gradient centrifugation. A distinct pattern of heterogeneity was determined on the basis of its harvesting time and biochemical criteria. Supernatants from cells cultured for 1 day contained a single peak of MIF activity with an isoelectric point of 4.3 to 5.2, an apparent m.w. of 23,000, and a density of 1.314 g/ml, the same as the density of the marker protein, 125I-HSA (1st day pH 5-MIF). Furthermore, this species of human MIF was sensitive to treatment with trypsin, strongly suggesting its being a protein, but not to treatment with neuraminidase and corresponds therefore to guinea pig pH 5-MIF. However, when 2nd day supernatants were analyzed under the same conditions, 2 MIF species were found. One species with an isoelectric point of 2.4 to 3.3 had an apparent m.w. of 65,000 (2nd day 3-MIF). The other species with an isoelectric point of 4.3 to 5.6 had an apparent m.w. of 23-43,000 (2nd day pH 5-MIF). Upon centrifugation in CsCl density gradients, the density (rho 25 of 1.314 to 1.414 g/ml) of both species was found to be greater than that of the pure protein, 125I-HSA. In addition, both species were chymotrypsin and neuraminidase sensitive but not trypsin sensitive, further suggesting their glycoprotein nature.

Dissociation of human interferon-gamma-like activity from migration-inhibition factor.

Supernatants harvested from concanavalin A-stimulated human peripheral mononuclear cells after 24 hr of incubation contain one interferon species similar to human interferon-gamma (IFN-gamma) with a pI of 4.6-5.3 (first day pH 5 IFN-gamm). In contrast, during the subsequent 24 hr of incubation two species with properties of IFN-gamma are produced with pI of 3.6-4.0 (second day pH 4 IFN-gamma) and 4.6-5.6 (second day pH 5 IFN-gamma), respectively. First day pH 5 IFN-gamma and second day pH 5 IFN-gamma have been found to differ on the basis of trypsin sensitivity. This pattern of polymorphism is similar to the pattern previously described for human migration-inhibitory factor (MIF) which can be separated into first day pH 5 MIF, second day pH 3 MIF, and second day pH 5 MIF. However, IFN-gamma-like species can be differentiated from MIF biochemically and antigenically. Fractions with second day pH 4 IFN-gamma have no MIF activity and fractions with second day pH 3 MIF contain no IFN activity. In addition, first and second day pH 5 MIF, which also contain IFN-gamma activity, can be separated from the latter by precipitation as well as neutralization with polyclonal and monoclonal anti-human MIF antibodies.

Recombinant migration inhibitory factor induces nitric oxide synthase in murine macrophages.

A recombinant form of migration inhibitory factor (MIF) obtained from COS-1 cells transfected with a cDNA library from a human T cell hybridoma is able to activate, in a dose-dependent manner, murine macrophages to express nitric oxide (NO) synthase and to produce high levels of NO in vitro. The time course of the induction of NO synthase is similar to that produced by the IFN-gamma. Enzyme activity peaks at 24 h and is undetectable by 72 h. MIF can synergize with IFN-gamma in the induction of NO synthesis, and the induction of NO synthase by both MIF and IFN-gamma is sensitive to inhibition by dexamethasone. However, unlike IFN-gamma-induced NO generation, MIF is sufficient for the induction of the enzyme, does not synergize with LPS, and is highly sensitive to inhibition by transforming growth factor.

The major binding protein of the interferon antagonist sarcolectin in human placenta is a macrophage migration inhibitory factor.

The interferon antagonist and growth promotor sarcolectin has affinity for negatively charged carbohydrates. Isolation of cellular binding proteins will be a step to elucidate its physiological significance. Thus, resin-immobilized sarcolectin was employed as affinity ligand for chromatographic fractionation of extract from human placenta. Elution with 0.1 M NH4OH or with 0.1 M N-acetylneuraminic acid and 1 M NaCl resulted primarily in purification of a protein of molecular mass of about 12 kDa according to gel electrophoretic analysis under denaturing conditions in the presence or absence of reductive agent and 12,470 Da by laser desorption mass spectrometry. The native molecular mass, assessed by gel filtration, is approximately 28 kDa. No evidence for detectable post-translational modification by glycosylation was provided by treatment with N-glycosidase F or sialidase and subsequent electrophoretic analysis. The N-terminal sequence of the major sarcolectin-binding protein is identical to that deduced from the cDNA sequence of a human macrophage migration inhibitory factor (MIF), starting from its third amino acid, over the determined stretch of 22 amino acids. Comparison of the calculated molecular mass of 12,221 of this factor to the experimentally determined value of 12,470 excludes any extensive modification of the protein. The sarcolectin-binding protein reduces macrophage migration at a concentration of 100 ng/ml in MIF assays. Recombinant migration inhibitory factor and purified sarcolectin-binding protein reacted equally well with anti-MIF antibody in immunoblot analysis and in assays to block binding to sarcolectin. Binding of biotinylated sarcolectin, too, is nearly identical for the two protein preparations. It is optimal in the range pH 7-9 and is markedly impaired by increasing ionic strength. Chemical modification with group-specific reagents revealed that the integrity of carboxyl groups of the sarcolectin-binding protein and of lysine/arginine groups of sarcolectin are primarily important to maintain binding capacity. In addition to contribute to the understanding of the functional significance of sarcolectin this result provides a convenient procedure to purify a lymphokine.

Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor.

By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a full-length cDNA encoding mouse GIF was isolated. A cDNA clone encoding human GIF was isolated from cDNA libraries of a GIF-producing human T-cell hybridoma by using mouse GIF cDNA as a probe. The cDNAs encode a putative 12.5-kDa peptide of 115 amino acids. Northern blot analysis demonstrated a single, 0.6-kb transcript. Polyclonal rabbit antibodies against the Escherichia coli-derived recombinant 13-kDa peptide bound hybridoma-derived GIF. Although the peptide did not contain a signal peptide sequence, transfection of the cDNA into COS-1 cells resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF. However, expression of a chimeric cDNA encoding a fusion protein consisting of the N-terminal pro region of calcitonin precursor and human GIF and cotransfection with furin cDNA to allow intracellular cleavage of the fusion protein resulted in secretion of 13-kDa peptide that was comparable to hybridoma-derived GIF in its bioactivity. Both the 13-kDa peptide and GIF bioactivity in the transfected COS-1 supernatant bound to a monoclonal antibody against hybridoma-derived human GIF. These results indicate that the 13-kDa peptide represents recombinant GIF, but posttranslational modification of the peptide is important for generation of the bioactivity. The GIF cDNA had high homology with the cDNA encoding macrophage migration inhibitory factor. However, the recombinant GIF failed to inhibit migration of human monocytes, and recombinant human macrophage migration inhibitory factor did not have GIF bioactivity.