Chronically HIV-1-infected monocytic cells induce apoptosis in cocultured T cells.

We have previously developed a human macrophage hybridoma model system to study the effect of HIV-1 infection on monocytic function. Upon coculture of one chronically (35 days postinfection) HIV-1-infected human macrophage hybridoma cell line, 43HIV, there was a dose-dependent decrease in the viability of cocultured Ag-stimulated T cells associated with an increase in DNA strand breaks. Enhanced apoptosis was determined by labeling with biotinylated dUTP and propidium iodide, increased staining with annexin V, increased side light scatter and expression of CD95, and decreased forward light scatter and expression of Bcl-2. There was also increased DNA strand breaks as determined by propidium iodide staining in unstimulated T cells cocultured with 43HIV and in T cells stimulated with anti-CD3 mAb and PHA. Pretreatment with 5145, a human polyclonal anti-gp120 Ab that recognizes the CD4 binding region, as well as with an anti-Fas ligand mAb blocked apoptosis in CD4+ T cells but not in CD8+ T cells. A soluble factor with a Mr below 10,000 Da was defined that induced apoptosis in CD4+ and CD8+ T cells and B cells. SDS-PAGE analysis of the active fractions revealed a band of 6000 Da that, after electroelution, had proapoptotic activity. The pI of the activity was estimated to be between 6.5 and 7.0. In conclusion, chronically HIV-1-infected monocytic cells induce apoptosis in bystander-, Ag-, anti-CD3-, and mitogen-stimulated T cells by multiple factors, which may contribute to the depletion of lymphocytes induced by HIV-1.

Absence of neutralizing antibodies to interferon in condyloma acuminata and cancer patients treated with natural human leukocyte interferon.

Human leukocyte-derived interferon-alpha (IFN-alpha n3) was used to treat condyloma acuminata patients in a double-blind placebo-controlled clinical study. The incidence of antibody formation to IFN-alpha was evaluated in matched patient sera from the control placebo and the IFN-alpha n3 treatment groups. Sera from IFN-alpha n3-treated phase I cancer patients and untreated healthy donors were also evaluated. Three sensitive assay methods (ELISA, competitive immunoradiometric, and antiviral neutralization) were used in these evaluations. The overall levels of detectable binding anti-IFN-alpha antibodies in the patients were similar to those of the normal donors. No neutralizing antibodies were generated in the patients after repeated treatment with natural IFN-alpha n3.

Serum activity that confers acid lability to alpha-interferon in systemic lupus erythematosus: its association with disease activity and its independence from circulating alpha-interferon.

We conducted a longitudinal evaluation of a patient with systemic lupus erythematosus who constitutively exhibited elevated levels of circulating alpha-interferon (alpha-IFN). This study demonstrated that the serum levels of an activity that renders the endogenous alpha-IFN acid labile are positively correlated with disease activity. This IFN acid lability-inducing activity can also be found in the sera of systemic lupus erythematosus patients who have active disease but who do not have circulating alpha-IFN.

Endothelial interleukin-8: a novel inhibitor of leukocyte-endothelial interactions.

Certain inflammatory stimuli render cultured human vascular endothelial cells hyperadhesive for neutrophils. This state is transient and reversible, in part because activated endothelial cells secrete a leukocyte adhesion inhibitor (LAI). LAI was identified as endothelial interleukin-8 (IL-8), the predominant species of which is an extended amino-terminal IL-8 variant. At nanomolar concentrations, purified endothelial IL-8 and recombinant human IL-8 inhibit neutrophil adhesion to cytokine-activated endothelial monolayers and protect these monolayers from neutrophil-mediated damage. These findings suggest that endothelial-derived IL-8 may function to attenuate inflammatory events at the interface between vessel wall and blood.

Interferon alpha associated with systemic lupus erythematosus is not intrinsically acid labile.

The physicochemical properties of apparently acid-labile IFN-alpha from patients with SLE have been studied. The antigenicity, apparent molecular size, and isoelectric point of SLE IFN-alpha are indistinguishable from those of conventional, previously characterized, acid-stable subspecies of IFN-alpha. However, after partial purification by anion-exchange chromatography, SLE IFN-alpha no longer exhibits acid lability, suggesting that other plasma factor(s) are responsible for the acid lability of SLE IFN-alpha. Addition of SLE plasma, but not normal plasma, to conventional acid-stable IFN-alpha renders the exogenous IFN-alpha acid labile. Preliminary results demonstrate that an acid-dependent IFN-inactivating activity can be partially purified from SLE plasma by anion-exchange chromatography.

Enhancement of cAMP levels and of protein kinase activity by tumor necrosis factor and interleukin 1 in human fibroblasts: role in the induction of interleukin 6.

Although tumor necrosis factor (TNF) and interleukin 1 (IL-1) affect many cell functions, the molecular mechanisms of TNF and IL-1 action are not understood. Our present study shows that exposure of human FS-4 fibroblasts to TNF or IL-1 caused a rapid accumulation of intracellular cAMP and an increase in protein kinase activity. Intracellular cAMP levels peaked 3-5 min after the addition of TNF or IL-1 and returned to basal level by 15 min. Increased phosphorylation of histone HII-B protein was demonstrated with extracts prepared from TNF- or IL-1-treated cells, suggesting an increase in cAMP-dependent protein kinase activity. No evidence was obtained for protein kinase C activation in TNF-treated FS-4 cells. TNF, IL-1, and forskolin all stimulated interleukin 6 (IL-6) mRNA levels in FS-4 cells. The protein kinase inhibitor H-8, inhibiting preferentially cAMP-dependent kinase activity, reduced forskolin-stimulated IL-6 mRNA induction more strongly than TNF- or IL-1-driven IL-6 mRNA induction. These results suggest that activation of cAMP-dependent protein kinase by TNF and IL-1 is important in some actions of these cytokines. In addition, our data on IL-6 induction by TNF and IL-1 suggest that other, yet unidentified, signal transduction mechanisms contribute to TNF and IL-1 actions on gene expression in human fibroblasts.

Toxicity of enzymically-oxidized low-density lipoprotein.

Intravenous injection of cholesterol oxidase into hyperlipidemic rabbits in which aortic atheromatous lesions have been induced by dietary means is lethal within hours, whereas injection of the same enzyme into normal rabbits has no visible adverse effect. The lethal effect of the enzyme is explicable by the finding that injection of cholesterol-oxidase treated low-density lipoprotein kills normal rabbits, in contrast to untreated low-density lipoprotein which does not. Enzymically oxidized low-density lipoprotein was also found to be cytotoxic for two human cell lines and for cultured bovine aortic endothelial cells. We suggest that in vivo enzymic conversion of low-density lipoprotein cholesterol to low-density lipoprotein cholestenone may possibly play a role in the initiation of atheromatous lesions in humans.

Interferon-gamma enhances expression of cellular receptors for tumor necrosis factor.

Incubation of several human tumor cell lines with human interferon-gamma (IFN-gamma) increased the specific binding of subsequently added 125I-labeled recombinant human tumor necrosis factor (TNF). A similar increase in TNF binding was seen in murine L929 cells after incubation with murine IFN-gamma, but not after incubation with human IFN-gamma. Increased TNF binding to cells incubated with IFN-gamma was due to an increase in the number of TNF receptors, with no demonstrable change in binding affinity. In one out of two human cell lines tested, IFN-alpha and IFN-beta also produced increased TNF binding, albeit with a lower efficacy than IFN-gamma. A maximal increase in TNF binding was seen after about 6 to 12 hr of incubation with IFN. Increased TNF binding due to enhanced TNF receptor expression may contribute to the enhancement of TNF cytotoxicity seen in some tumor cell lines after INF treatment. Modulation of TNF receptor expression by IFN may also influence other biological activities of TNF.

Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells.

Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 125I and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 125I-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 X 10(-10) M and 3.2 X 10(-10) M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 37 degrees C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF.

Cloning and expression of a novel variant of human interferon-gamma cDNA.

A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.

Monoclonal antibodies as structural probes for oligomeric human interferon-gamma.

Monoclonal antibodies (MAb) B1 and B3, specific for human interferon-gamma (IFN-gamma) failed to immunoprecipitate heat-inactivated human IFN-gamma in solution. However, both MAb retained some reactivity with denatured IFN-gamma immobilized on vinyl plates. The two MAb have been employed in a sensitive immunoradiometric assay (IRMA). In this IRMA one MAb was bound to polystyrene beads and used as immunoadsorbent. The second MAb, labeled with 125I, was used as the tracer to quantitate the amount of IFN-gamma bound to the immobilized MAb. Addition of unlabeled MAb B1 did not inhibit the binding of 125I-labeled MAb B3 (and vice versa), indicating that the two MAb react with two different and nonoverlapping epitopes. Yet, when the same MAb was used in IRMA as both immunoadsorbent and tracer, the amount of labeled MAb bound to a given concentration of natural or E. coli-derived recombinant human IFN-gamma was very similar as with two different MAb, indicating that a single IFN-gamma molecule must have two or more identical binding sites for each of the two MAb. These findings show that biologically active natural and recombinant human IFN-gamma exist in oligomeric form.

Cytolytic activity of interferon-gamma and its synergism with 5-fluorouracil.

Highly purified natural or recombinant human immune interferon (IFN-gamma) was found to be directly cytolytic to certain tumor cell lines in vitro. Out of 5 human tumor cell lines and one normal fibroblast line tested, the colon adenocarcinoma line HT-29 and the rhabdomyosarcoma line A673 were highly sensitive to cytolysis by interferon, as determined by 125I-iododeoxyuridine release in a 72 h microcytotoxicity assay. Cytolysis was marked at IFN-gamma concentrations of less than I U/ml, and it reached a near-maximal level at 6.4 U/ml. A synergistic cytolysis on HT-29 cells of IFN-gamma and 5-fluorouracil (5-FU) was observed at 5-FU concentrations ranging from 64 to 640 micrograms/ml. In contrast, no synergism was observed between IFN-gamma and mitomycin C. The direct cytolytic activity and synergistic cytolysis with 5-FU of the IFN-gamma preparations used in the present study were abolished completely by treatment with a neutralizing monoclonal antibody specific for human IFN-gamma.

Three molecular weight forms of natural human interferon-gamma revealed by immunoprecipitation with monoclonal antibody.

Immune interferon (IFN-gamma), endogenously labeled with [35S]methionine, was produced in human peripheral blood lymphocyte cultures stimulated with 12-O-tetradecanoylphorbol-13-acetate and phytohemagglutinin. 35S-IFN-gamma, immunoprecipitated from the crude culture fluid with a monoclonal antibody, was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three monomeric forms with molecular weights of 25,000, 20,000, and 15,500, which we designate IFN-gamma I, II, and III, respectively. IFN-gamma I was the most, and IFN-gamma III the least, abundant in both immunoprecipitated 35S-IFN-gamma and chromatographically purified IFN-gamma preparations. Changes in the molecular size of the monomeric forms after glycosidase treatment suggested that IFN-gamma I contains more carbohydrate than IFN-gamma II, and that IFN-gamma III may not be glycosylated at all. Hence, the differences in the carbohydrate contents are likely to be the major cause of the molecular size heterogeneity of IFN-gamma I, II, and III.

Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin.

Crude preparations of interferon (IFN)-gamma derived from human peripheral blood leukocyte (PBL) cultures induced with 12-O-tetra-decanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA) were more cytotoxic to HeLa cells than partially purified nautral or highly purified recombinant human IFN-gamma preparations. Conditioned media from PBL cultures contained, in addition to IFN-gamma, a mixture of cytotoxins, including classic lymphocyte-derived lymphotoxin (LT), and a TPA-induced cytotoxic activity produced by the adherent cell population (presumably monocytes). These two types of cytotoxins, indistinguishable in the mouse L929 cell LT assay, could be differentiated by an antiserum prepared against LT derived from the B lymphoblastoid cell line RPMI 1788. This antiserum neutralized lymphocyte-derived classic LT but failed to neutralize the activity of the monocyte-derived cytotoxin. Processing of conditioned media by sequential chromatography on silicic acid, Con A-Sepharose, and DEAE-Sephacel failed to separate IFN-gamma from the LT activity. However, this procedure did remove the monocyte-derived cytotoxic activity present in the original starting material, leaving predominantly classic LT. This LT showed a slightly basic isoelectric point (pI 7.6) which partially overlapped the more basic pI range of IFN-gamma. The two lymphokine activities also could not be completely separated by fast protein liquid chromatography or molecular sieve chromatography. LT in these partially purified preparations was associated with a protein having an apparent molecular weight of 58,000 on gel filtration. This form dissociated partially into a 20,000 mol wt species after denaturation with 0.1% NaDodSO4. IFN-gamma could be selectively removed from preparations containing both IFN-gamma and LT with the aid of monoclonal antibody to IFN-gamma. The addition of purified LT to purified E. coli-derived recombinant human IFN-gamma resulted in a marked synergistic enhancement of cytotoxicity for HeLa cells.

Activation of human monocyte cytotoxicity by natural and recombinant immune interferon.

Human T cell hybridomas were established by fusion of SH9 cells, the 6-thioguanine-resistant mutant line of human T lymphoma Hut 102-B2, with concanavalin A-stimulated human peripheral blood lymphocytes. Hybridoma line L38 produced a macrophage activating factor (MAF) with the ability to activate human peripheral blood monocytes to show enhanced cytotoxicity against human colon adenocarcinoma HT-29 cells in a 72-hr 125iododeoxyuridine-release assay. The L38 line was then cloned by the limiting dilution technique and two sublines, L38B and L38D, were found to produce high levels of MAF constitutively. Interferon activity was also detected in L38B and L38D supernatants. When interferon activity was neutralized with specific antiserum to purified human immune interferon (IFN-gamma), MAF activity was abrogated. To confirm that the MAF activity is indeed due to IFN-gamma, IFN-gamma was purified from the culture supernatant of another human T cell hybridoma, L265K2, a cell line known to produce high levels of IFN-gamma. Two highly purified IFN-gamma fractions with m.w. of 20,000 and 25,000, respectively, were obtained by NaDodSO4/polyacrylamide gel electrophoresis (SDS-PAGE). Similar fractions were obtained from IFN-gamma derived from human peripheral blood lymphocyte (PBL) cultures induced with 12-0-tetradecanoylphorbol-13-acetate (TPA) and phytohemagglutinin (PHA). In comparison, Escherichia coli-derived recombinant human IFN-gamma separated by SDS-PAGE yielded two major active fractions with m.w. of 17,000 and 34,000. With all three types of preparations, a close correlation was found between the presence of IFN-gamma activity demonstrable in an antiviral assay and MAF activity in individual fractions. Substantial quantitative differences were observed in the ability of various human IFN to activate monocytes. Although no MAF activity was detected with IFN-alpha and IFN-beta at concentrations up to 200 U/ml, both natural and recombinant IFN-gamma showed marked MAF activity at concentrations as low as 0.3 to 1 U/ml.

Stimulation of lymphokine production by teleocidin, aplysiatoxin, and debromoaplysiatoxin.

Previous studies showed that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-gamma) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-gamma and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-gamma during a multistep purification procedure.

Effects of glycosidase treatment on the physicochemical properties and biological activity of human interferon-gamma.

Highly purified human interferon (IFN)-gamma was treated with a preparation of mixed glycosidases in order to evaluate the effect of carbohydrate depletion on its biological activity, isoelectric point, and molecular size. Glycosidase treatment did not reduce the antiviral activity of IFN-gamma in cultures of human fibroblasts and in bat lung cells. No antiviral activity was observed before or after treatment with glycosidases in pig, mink, bovine, murine, and monkey cells. The degree of neutralization of IFN-gamma activity with specific antibody was also not significantly affected by glycosidase treatment. Several components of IFN-gamma activity were resolved by nonequilibrium pH gradient electrophoresis, with major peaks of activity at pI 8.5 and 8.7. Glycosidase treatment of IFN-gamma resulted in a reduced charge heterogeneity and a higher pI of 9.3. 125I-labeled IFN-gamma was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two bands with molecular weights of 25,000 and 20,000. Glycosidase treatment reduced the apparent molecular weight of these bands to 18,500 and 16,000, respectively. The results suggest that both the Mr = 25,000 and 20,000 bands, thought to be monomeric forms of IFN-gamma, are glycosylated.

Human interferon-gamma is internalized and degraded by cultured fibroblasts.

Human interferon-gamma (IFN-gamma) binds specifically and with high affinity to receptors on the surface of cultured fibroblasts (GM-258). At 37 degrees C about 50% of the receptor-bound IFN-gamma was rapidly internalized (t 1/2 = 4-5 min) by these cells. Following an initial lag of 15-30 min, internalized IFN-gamma was continuously degraded over a period of at least 8 h. The total uptake of IFN-gamma over this time period was found to exceed by 5 times the number of occupied IFN receptors present on the surface of these cells, suggesting that either there is a large intracellular pool of IFN-gamma receptors, or that receptors are recycled during the course of incubation. Cycloheximide (100 micrograms/ml) inhibited uptake only after the first 2 h of incubation and then only moderately. It is therefore unlikely that de novo receptor synthesis plays a major role in the observed uptake process. Both sodium azide (15 mM) and methylamine (20 mM) inhibited both the uptake and degradation of IFN-gamma at all times up to 6 h. While uptake was only slightly reduced in the presence of chloroquine (25 microM), degradation was markedly inhibited, suggesting that degradation occurs intracellularly, probably within lysosomes.