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.

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.

Molecular weight of human gamma interferon is similar to that of other human interferons.

The molecular weight (as determined by molecular sieve chromatography) of human gamma interferon, formerly referred to as immune or type II interferon, is between 40,000 and 70,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gamma interferon activity was recovered mainly from two regions of the gels corresponding to molecular weights of 20,000 and 25,000. The results suggest that in native form human gamma interferon may be aggregated.

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.

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.

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.

Reconstitution of native human hemoglobin from separated globin chains and alloplex intermediates.

A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free alpha- and beta-globin chains (alpha degrees, beta degrees) and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the allosteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at high dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For alpha-globin, the yield is about 50% because of incomplete combination with the heme-containing beta chain. For beta-globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.

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.

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.

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.

Specific binding of 125I-human interferon-gamma to high affinity receptors on human fibroblasts.

Highly purified human interferon-gamma (IFN-gamma) was iodinated with 125I-Bolton-Hunter reagent to a specific activity of 34 microCi/micrograms of protein. After iodination, molecular sieve chromatography was used to isolate fractions containing IFN-gamma at approximately 80-90% radioactive purity. This preparation of 125I-IFN-gamma bound specifically to high affinity binding sites on human GM-258 fibroblasts. Scatchard analysis of binding data revealed the presence of 2400 binding sites/cell, each binding with an apparent Kd of 1.5 X 10(-10) M. Binding was competitively inhibited in the presence of unlabeled IFN-gamma and, to a lesser degree, by IFN-beta, but not by IFN-alpha. Neutralizing antibodies specific for IFN-gamma efficiently inhibited the specific binding of 125I-IFN-gamma to cells. We conclude that the specific high affinity binding site is the receptor for IFN-gamma.

Studies on catalytic properties of purified high molecular weight pancreatic lipase.

The properties of the 500-fold purified high-molecular-weight lipase have been studied. The rate of hydrolysis of the triglycerides decreases with increasing fatty acid chain length. The lipolytic activity also increases with increase in unsaturation in the fatty acyl moiety. Diglycerides are hydrolyzed at more than twice the rate for triglycerides while monoglycerides are not hydrolyzed. Methyl esters are generally hydrolyzed at a higher rate which increases with increasing chain length of the fatty acid but the enzyme does not act on phospholipids. Emulsifying agents such as Tween 20, gum arabic, and albumin increase the rate of hydrolysis. Metal ions such as Hg2+, Zn2+, Cu2+, and Fe2+ strongly inhibit the lipolytic activity of the high-molecular-weight lipase while Ca2+ or Mg2+ by themselves show no stimulating effect. Treatment of the high-molecular-weight lipase with P-chloromercurybenzoate inhibits hydrolytic activity by 70% while iodoacetic acid has no effect.

Characteristics of human lymphoblastoid (Namalva) interferon.

Interferon derived from the human lymphoblastoid cell line, Namalva, was fractionated by antibody affinity chromatography into two antigenically distinct interferon subspecies. At least 13% of the total Namalva interferon activity possessed the F antigenic determinant found on human interferon derived from fibroblast cultures, while the bulk of the Namalva interferon activity had the Le antigenic determinant characteristic for human leukocyte interferon. The separated Le and F subspecies of Namalva interferon differed in the degree of their heterospecific activities on bovine cells. The Le moiety resembled crude leukocyte interferon in that it was highly active in bovine cells. The F component of Namalva interferon showed a lower degree of activity in bovine cells, thus resembling crude fibroblast interferon. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by isoelectric focusing, crude Namalva interferon qualitatively resembled crude leukocyte interferon.

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.

Pharmacokinetic properties of human fibroblast and leukocyte interferon in rabbits.

When rabbits were given intramuscular injections of the same quantities of human leukocyte or fibroblast interferons, the former produced moderately higher levels of circulating interferon. Fibroblast interferon was not cleared faster from circulation, nor was direct inactivation by rabbit blood responsible for this difference.

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.

Partial purification and characterization of human gamma (immune) interferon.

Human gamma (immune) interferon (IFN-gamma) was produced in lymphocyte cultures stimulated with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) and purified phytohemagglutinin. Physicochemical analysis showed that human IFN-gamma is a glycoprotein with an isoelectric point around 8.6 and an apparent molecular weight of 58,000 +/- 3000. A purification process for IFN-gamma was developed consisting of sequential chromatographic separations on controlled-pore glass, concanavalin A-Sepharose, and Bio-Gel P-200. This purification process resulted in an increase in specific activity from about 10(4) (crude culture fluid) to an estimated 10(7) units per mg of protein with a cumulative recovery of about 40% of the IFN activity.

Partial characterization of gamma (immune) interferon mRNA extracted from human lymphocytes.

gamma (immune) interferon (IFN-gamma) was induced in cultures of fresh human lymphocytes by combined treatment with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate, TPA) and the T cell mitogen phytohemagglutinin (PHA). Compared to the IFN-gamma yields obtained with PHA induction alone, the inclusion of TPA caused a significant enhancement of IFN-gamma production. Poly(A)-containing mRNA was isolated from mononuclear cells induced with TPA and PHA. Injected into Xenopus laevis oocytes, this mRNA preparation gave rise to IFN activity with characteristic properties of human IFN-gamma. Sucrose density gradient centrifugation analysis showed that IFN-gamma mRNA sedimented at 15 S, suggesting that it contains a total of about 1400 nucleotides.