Interferon-gamma-induced degradation of tryptophan by human cells in vitro

Several human cells were investigated for their ability to degrade tryptophan and to synthesize neopterin upon induction by interferon-gamma (500 units/ml for 48 h). Concentrations of tryptophan, kynurenine, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, 7,8-dihydroneopterin and neopterin were assessed in the culture supernatants by HPLC. Fibroblasts, A-22 arachnoidea, HK-2351 scalp, T-2346 meningeom and HeLa cervical carcinoma cells but not HL-60 promyelocytic leukaemia cells were found to degrade tryptophan upon induction by interferon-gamma. Tryptophan is converted to kynurenine by fibroblasts, A-22 arachnoidea and HK-2351 scalp cells and to kynurenine and anthranilic acid by HeLa cervical carcinoma and T-2346 meningeom cells. Kynurenine and anthranilic acid always make up more than 82% of the tryptophan degraded. None of these cells synthesizes 3-hydroxyanthranilic acid, 3-hydroxykynurenine, 7,8-dihydroneopterin or neopterin. Human macrophages form 3-hydroxyanthranilic acid and neopterin, but not 3-hydroxykynurenine, beside kynurenine and anthranilic acid upon activation by interferon-gamma. These data indicate that several human cells can be induced by interferon-gamma to degrade tryptophan. The interferon-gamma induced synthesis of 3-hydroxyanthranilic acid and neopterin, however, appears to be restricted to human macrophages. A hypothesis explaining these findings is presented.     

Biosynthesis and metabolism of pterins in peripheral blood mononuclear cells and leukemia lines of man and mouse

The cellular origin and the control of neopterin release associated with immune stimulation was studied in cell cultures. Using purified human mononuclear cells, the intracellular change in concentrations of GTP and pterins was measured under various kinds of stimulation. Three enzymes involved in tetrahydrobiopterin biosynthesis, i.e. GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase, were also determined. Human macrophages stimulated with culture supernatant from activated T-lymphocytes were the main producers of neopterin. In these cells, GTP cyclohydrolase I activity was elevated due to high GTP levels and therefore neopterin accumulated. Human macrophages lack 6-pyruvoyl tetrahydropterin synthase activity. Exogenous tetrahydrobiopterin added to the culture medium of stimulated T cells and macrophages suppressed the elevation of GTP cyclohydrolase I activity and neopterin concentration, but not the elevation of intracellular GTP. Stimulation of macrophages with recombinant human interferon-gamma and neutralization of the effect of T cell supernatants by addition of a monoclonal antibody specific for human interferon-gamma showed that immune interferon induced the alterations in GTP cyclohydrolase I activity and neopterin concentration. In the human macrophage line U-937 and in the leukemia line HL-60, no GTP cyclohydrolase I activity or intracellular pterins were detected, but high levels of GTP. In mouse mononuclear cells, no neopterin was detected, but biopterin and pterin. After stimulation, biopterin was elevated in the same way as neopterin in human mononuclear cells. This is explained by the different regulation of the rate-limiting steps of tetrahydrobiopterin biosynthesis in man and in mouse. These results suggest that neopterin is an unspecific marker for the activation of the cellular immune system.     

St. John's wort (Hypericum perforatum) counteracts cytokine-induced tryptophan catabolism in vitro

St. John's wort (Hypericum perforatum) is an ancient folk remedy that has antiviral and antibacterial properties. Anti-inflammatory effects of the plant have been described and the application of H. perforatum extract as an effective antidepressant is well established. In this study we assayed the effect of H. perforatum extract on cytokine-induced tryptophan degradation in human peripheral blood mononuclear cells. Simultaneously, changes in the production of the immune activation marker neopterin were monitored. Both these biochemical pathways are triggered by interferon-gamma. Our results show that extracts of H. perforatum strongly down-regulate mitogen-mediated tryptophan degradation in a dose-dependent manner. This effect seems to be based on a suppressive activity of H. perforatum on activated immunocompetent cells, resulting in a diminished production of interferon-gamma. In line with this finding, neopterin synthesis was strongly down-regulated by the plant extract. Our results suggest that the reduction of tryptophan degradation by H. perforatum might be important in the action of the plant as an antidepressant.     

Characteristics of interferon induced tryptophan metabolism in human cells in vitro

Interferon-gamma-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal human fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by interferon-gamma. Five of these ten formed only kynurenine (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts), three formed kynurenine and anthranilic acid (U 138 MG, glioblastoma; SK-HEP-1, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthranilic acid and 3-hydroxyanthranilic acid). Interferon-gamma regulated only the activity of indoleamine 2,3-dioxygenase. All other enzyme activities detected were independent of interferon-gamma, as shown by the capacity of the cells to metabolize L-kynurenine or N-formyl-L-kynurenine. Increasing the extracellular L-tryptophan concentration resulted in a marked induction of tryptophan degradation by macrophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular L-tryptophan concentration was increased 2-fold with SK-N-SH, T 24 and J 82, 4-fold with A 431 and A 549 and 10-fold with U 138 MG and SK-HEP-1. The activity was unaffected by extracellular L-tryptophan with dermal fibroblasts and A 498. Though interferon-gamma was the most potent inducer of tryptophan metabolism, interferon-alpha and/or -beta showed small but distinct action on some of the cells. In all cells which reacted to interferon-gamma by enhanced expression of class I and/or class II major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,3-dioxygenase is a common feature of interferon-gamma action, that the extent of this induction is influenced by extracellular L-tryptophan concentrations and that indoleamine 2,3-dioxygenase is the only enzyme in the formation of 3-hydroxyanthranilic acid from tryptophan which is regulated by interferon-gamma.     

Establishment of an interleukin 2-dependent T cell line derived from a patient with severe aplastic anemia, which inhibits in vitro hematopoiesis

Circulating mononuclear cells from a patient developing severe aplastic anemia during the course of non-A, non-B hepatitis were found to be virtually entirely composed of in vivo activated suppressor T cells (Ia+T8+). These cells were used to establish a new permanent cell line, termed SMAA, by using phytohemagglutinin, Ebstein-Barr virus-transformed irradiated B cells, allogeneic irradiated peripheral blood mononuclear cells, and recombinant interleukin 2 to investigate the relationship of aplastic anemia-derived circulating T cells to bone marrow failure. SMAA cells, now in continuous culture for more than 9 mo, were shown to inhibit proliferation of purified myeloid progenitors and their differentiation into early and late appearing neutrophil and eosinophil colonies by 90%, whereas monocyte colonies were much less affected. Similarly, growth of erythroid colonies and bursts was almost completely inhibited, as was anti-mu-induced B cell proliferation and lectin-induced T cell proliferation. This inhibition of hematopoiesis was mediated by the release of a soluble factor that was sensitive to acid (pH 2), heat (56 degrees C), and trypsin. Monoclonal and polyclonal antibodies to interferon-gamma could abrogate the inhibitory effects of SMAA supernatant, but more than 10(4) neutralizing U/ml had to be added. The effects of SMAA could be duplicated by adding 10(4) U/ml of purified recombinant interferon-gamma to colony and proliferation assays. The concentration of interferon-gamma in SMAA supernatant was estimated to be greater than 3 X 10(3) National Institutes of Health reference U/ml by immunoradiometric assay. These results demonstrate that some patients with aplastic anemia have circulating T cells that are capable of prolonged in vitro secretion of interferon-gamma causing severe inhibition of in vitro hematopoiesis, and these cells can be expanded into permanent lines for studies on their regulatory properties.     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

Malaria Antigens Stimulate Neopterin Secretion by PBMC and U937 Cells

Lysates of Plasmodium falciparum parasitized human erythrocytes stimulate U937 cells to secrete neopterin during a 48 hr co-culture period. Neopterin secretion by U937 cells was enhanced by the addition of human interferon gamma (IFN-γ). Several P. falciparum antigens, ‘FC27’ (a synthetic ‘S’ antigen), Ag16 (a recombinant ‘S’ antigen) and Ag44/RHOP3 (a recombinant merozoite rhoptry protein), also activated U937 cells to neopterin secretion and produced a similar effect on peripheral blood mononuclear cells (PBMC) from 2 of 3 normal healthy donors cultured with the antigens for 7 days. Plasma from six Nigerian malaria patients contained high neopterin concentrations ranging from 5.06 to 14.17 ng/ml. This preliminary pilot study lends support for further investigation incorporating a larger number of malaria patients and further culture experiments with U937 cells and PBMC with the aim of defining the cause and source of the large quantities of plasma neopterin produced in this infection.     

Biologic-response-modifier-induced indoleamine 2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures

Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been augmented in human epithelial cell lines treated with human interferon-gamma (HuIFN-gamma). Several human biologic response modifiers, including HuIFN-gamma, HuIFN-beta, HuIFN-alpha, interleukin 2 (HuIL-2), and tumor necrosis factor alpha, have now been assessed for their ability to enhance tryptophan degradation in human peripheral blood mononuclear cell (PMC) cultures. PMC were isolated from normal donors, cultivated in RPMI 1640 medium containing [3H]tryptophan, and treated with individual biologic response modifiers. At various intervals, culture supernatants were removed, fractionated by reversed-phase high performance liquid chromatography, and radioactivity in resultant fractions was determined. Significantly increased amounts of tryptophan catabolites were observed after treatment with HuIFN-gamma, HuIFN-beta, HuIFN-alpha, and HuIL-2, but not human tumor necrosis factor alpha. Often, greater than 30% of available tryptophan was degraded by treated PMC cultures. Although antibodies to HuIFN-alpha, HuIFN-beta, and HuIFN-gamma specifically neutralized the induction of IDO activity in PMC by their respective HuIFN, only anti-HuIFN-gamma antibody also neutralized HuIL-2-induced IDO activity. Furthermore, T24 bladder carcinoma cells, in which IDO was induced by HuIFN-gamma but not by the other biologic response modifiers, were induced to degrade tryptophan by supernatants of HuIL-2-stimulated PMC cultures, but not by HuIFN-beta-stimulated PMC culture supernatants. Thus, whereas HuIL-2 indirectly induced IDO in PMC cultures by stimulating production of HuIFN-gamma, all cases of interferons appeared to induce IDO directly in PMC cultures.     

T cell replacing factor for steroids (TRF-S): a 40,000 dalton protein produced by a T4+ T cell

The induction of polyclonal immunoglobulin (Ig) synthesis by glucocorticosteroids (GCS) in human peripheral blood lymphocytes is dependent on both T cells and monocytes. T cells can be replaced by a cytokine, T cell replacing factor for steroids (TRF-S), which promotes GCS-induced Ig production. T cells produce the cytokine when cultured with intact monocytes, with 24 hr monocyte supernatants, or with small quantities (0.1 U/ml or more) of highly purified interleukin 1 (IL 1). TRF-S was produced by isolated T4+ cells, whereas isolated T8+ cells were unable to help GCS-induced Ig synthesis. High pressure liquid chromatography with a gel permeation column revealed a single locus of activity that corresponded to an apparent m.w. of 40,000. At the dilutions utilized in culture, supernatants containing optimal TRF-S activity (3 U/ml final concentration in culture) were found to have less than 0.2 U/ml (final concentration) of interleukin 2 (IL 2) activity. Neither recombinant IL 2 nor recombinant interferon-gamma (IFN-gamma) over a broad range of concentrations was able to reproduce the capacity of TRF-S to induce the development of Ig-secreting cells with GCS. Thus, we report that TRF-S is synthesized primarily by T4+ T cells, and that its production is stimulated by small concentrations of IL 1. The apparent m.w. of TRF-S is 40,000, and its biological activity is distinct from that of IL 1, IL 2, and IFN-gamma.     

Synovial Mononuclear Cells Consist with T Cells Which Produce High Levels of Tumor Necrosis Factor α

To determine whether synovial mononuclear cells include a population of tumor necrosis factor α-produeing T cells, we measured tumor necrosis α levels in culture supernatants of synovial mononuclear cells by ELISA and analyzed tumor necrosis α mRNA-positive cell frequencies. There were no significant differences in the spontaneous levels of TNF α between synovial mononuclear cells and peripheral mononuclear cells. The frequency of tumor necrosis factor α mRNA-positive cells in synovial mononuclear cells was higher than that of peripheral mononuclear cells. When stimulated with a superantigen, mononuclear cells from the synovial fluid of rheumatoid arthritis patients showed higher levels of tumor necrosis factor α production (1,035 ± 817 pg/ml) than did mononuclear cells from their peripheral blood (236 ± 180 pg/ml). In addition, we observed that a few T cell clones were resistant to superantigenic restimulation in vitro. We conclude that when these types of T cells persist in the synovium, they play a role in the development of rheumatoid arthritis via a mechanism involving tumor necrosis factor α production.     

Biosynthesis of the third component of complement in rat liver epithelial cell lines and its stimulation by effector molecules from cultured human mononuclear cells

Summary Rat liver epithelial cell lines, growing in a serum-supplemented medium, synthesize and secrete into the culture medium the third component of complement (C₃). We studied the regulation of C₃ production in this system. We found that human peripheral blood mononuclear leukocytes in culture released one or more soluble factors which stimulated rat liver epithelial cells to produce increased quantitites of C₃. This stimulting effect was strongly enhanced when the mononuclear cell cultures were treated with phytohemagglutinin, a T-lymphocyte mitogen. The factor(s) failed to enhance C₃ biosynthesis by rat dermal fibroblasts, which are known to produce this protein. This reveals a tissue-specific differential response between the fibroblasts and the liver epithelial cells. The physical and chemical characteristics, such as heat sensitivity, 2.8M ammonium sulphate precipitation, and lower activity after digestion by proteases unambiguously indicate that the effector molecules are proteins. When the crude supernatant of mononuclear leukocytes was fractionated by gel filtration, the stimulating factor(s) eluted as two peaks with apparent molecular weight of 25 to 60 and 15 to 20 kdalton, respectively. As to the cellular origin of the C₃-stimulating factor(s), several observations were made: (a) in separate cultures containing either T-cells or monocyte-enriched populations from the same sample of blood mononuclear cells, no activity was detected in the presence or absence of phytohemagglutinin, (b) conditioned media from each of these cultures could not substitute for the corresponding intact cell populations, and (c) the addition of purified T-cells to the monocyte-enriched population in the presence of phytohemagglutinin restored the production of the stimulating activity by the mixed culture. Finally, experiments were carried out to verify whether monokine interleukin 1 affects the hepatic C₃ biosynthesis. It was demonstrated that interleukin 1 enhanced this biosynthesis, but could not completely substitute for conditioned medium from stimulated mononuclear cells.     

Monitoring of immune activation using biochemical changes in a porcine model of cardiac arrest

In animal models, immune activation is often difficult to assess because of the limited availability of specific assays to detect cytokine activities. In human monocytes/macrophages, interferon-gamma induces increased production of neopterin and an enhanced activity of indoleamine 2,3-dioxygenase, which degrades tryptophan via the kynurenine pathway. Therefore, monitoring of neopterin concentrations and of tryptophan degradation can serve to detect the extent of T helper cell 1-type immune activation during cellular immune response in humans. In a porcine model of cardiac arrest, we examined the potential use of neopterin measurements and determination of the tryptophan degradation rate as a means of estimating the extent of immune activation. Urinary neopterin concentrations were measured with high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA) (BRAHMS Diagnostica, Berlin, Germany). Serum and plasma tryptophan and kynurenine concentrations were also determined using HPLC. Serum and urine neopterin concentrations were not detectable with HPLC in these specimens, whereas RIA gave weakly (presumably false) positive results. The mean serum tryptophan concentration was 39.0 +/- 6.2 micromol/l, and the mean kynurenine concentration was 0.85 +/- 0.33 micromol/l. The average kynurenine-per-tryptophan quotient in serum was 21.7 +/- 8.4 nmol/micromol, and that in plasma was 20.7 +/- 9.5 nmol/micromol (n = 7), which corresponds well to normal values in humans. This study provides preliminary data to support the monitoring of tryptophan degradation but not neopterin concentrations as a potential means of detecting immune activation in a porcine model. The kynurenine-per-tryptophan quotient may serve as a short-term measurement of immune activation and hence permit an estimate of the extent of immune activation.     

Lymphokine stimulation of human macrophage C2 production is partially due to interferon-gamma

Monocyte complement stimulator (MCS), a product of T lymphocytes, is defined by its ability to stimulate the synthesis and secretion of the second complement component (C2) by monocytes. Most macrophage-activating factor (MAF) activity present in lymphokine-rich culture supernatants has recently been found to be due to interferon-gamma (IFN-gamma). We therefore hypothesized that IFN-gamma may have MCS activity as well. We tested recombinant, E. coli-derived, human IFN-gamma (rIFN-gamma) for its effects on C2 production by adherent peripheral blood monocytes and U937 cells, a human monocytic cell line. Recombinant IFN-gamma in concentrations ranging from 0.1 to 300 U/ml (0.003 to 8.8 ng/ml) stimulates C2 production by both cell populations. Exposure of responding cells for at least 24 hr is required for maximal stimulation. To determine the contribution of IFN-gamma toward total MCS activity in crude lymphokine-rich supernatants, we employed a solid-phase immunoabsorption technique with the use of a monoclonal anti-IFN-gamma antibody. This technique removed all IFN-gamma detectable by a sensitive ELISA, but MCS activity was decreased by only 40 to 50%. Additionally, MCS activity of these supernatants did not correlate with IFN-gamma content as determined by ELISA. By using another method to eliminate IFN-gamma activity, acid dialysis destroyed all rIFN-gamma activity, as measured by stimulation of U937 C2 synthesis, but eliminated only 30 to 67% of MCS activity from crude lymphokine preparations. Thus IFN-gamma stimulates C2 production by monocytes and U937 cells and apparently accounts for some, but not all, MCS activity present in lymphokine-rich supernatants. Other lymphokines are present in such supernatants that also possess this activity.     

Regulation of hepatic acute phase protein synthesis by products of interleukin 2 (IL 2)-stimulated human peripheral blood mononuclear cells

Cancer patients injected with recombinant human IL 2 develop marked changes in serum concentrations of hepatic acute-phase proteins. To determine if this acute-phase response involves a change in the rate of hepatic protein synthesis and if it is due to a direct effect of IL 2 on hepatocytes, human hepatoma-derived hepatocytes (Hep-3B cells) were incubated in medium containing IL 2 or in culture supernatants from IL 2-activated human peripheral blood mononuclear cells (PBMNC). The rate of synthesis of two acute-phase proteins, complement protein factor B and albumin, was determined by the incorporation of a radiolabeled amino acid precursor into newly synthesized protein as measured by analytical gel electrophoresis of immunoprecipitates. IL 2 in concentrations from 1 to 1000 U/ml had no effect on the synthesis of factor B or albumin; conversely, there was a dose-dependent increase in the rate of synthesis of factor B and decrease in albumin synthesis mediated by culture supernatants of IL 2-activated PBMNC. The magnitude of the effect of acute-phase protein synthesis was dependent on the IL 2 concentration used for the activation of PBMNC. The rate of factor B synthesis increased approximately 4.0-fold in the presence of culture supernatants of PBMNC activated with either opsonized heat-killed Staphylococcus albus or with 1000 U/ml IL 2. Preincubation of the IL 2-activated PBMNC culture supernatants with an antiserum specific for recombinant IL 1-beta completely neutralized the capacity of the supernatants to stimulate factor B synthesis, whereas antisera specific for human IL 1-alpha or for tumor necrosis factor had no effect. These results indicate that the indirect effect of IL 2 on hepatic acute phase protein synthesis is mediated by IL 1-beta.     

T-helper 1 and 2 serum cytokine assay in chronic opioid addicts

There are a few studies with conflicting results on the effects of opioids on the functioning of immune system. This study was performed to investigate the in vitro production of interferon-gamma and interleukin-10 after antigenic stimulation of cells using whole blood from opioid addicts. Blood samples were taken from 20 chronically opioid-addicted persons, who voluntarily enrolled for detoxification (10 opium and 10 heroin addicts). Blood samples were also taken from 10 healthy individuals with no history of drug abuse as the control. Cell culture was performed in a whole blood culture assay. Diluted blood samples were stimulated with phytohemagglutinin or with lipopolysaccharide and the supernatants were collected to measure cytokine production. The results demonstrated a significant decrease in interferon-gamma production and an increase in interleukin-10 secretion in heroin addicts, relative to the control group (35.9+/-26.3 versus 110.2+/-60.3 pg/mL, p<0.01 and 71.8+/-28.4 versus 17.1+/-13.5 pg/mL, p<0.01, respectively), however the changes in these values in opium addicts were not significant compared to healthy individuals. The results could suggest that opioid addiction leads to a shift in the Th1/Th2 cytokine balance of peripheral CD4+ cells towards the Th2 response, and opioid addicts demonstrate reduced mitogenic responsiveness of lymphocytes relative to healthy individuals.     

The role of T cells in immunoglobulin class switching of specific antibody production system in vitro in humans

Only antibodies of the IgM class were produced in vitro by peripheral blood mononuclear cells stimulated with streptococcal carbohydrate. B cells of the peripheral blood mononuclear cells, however, synthesized both IgM and IgG class antibodies when combined with tonsillar T cells, suggesting that T cells inducing immunoglobulin class switching are present in the tonsils. Peripheral blood T cells also became capable of inducing B cells to produce IgG class antibodies when the T cells were incubated with antigen-pulsed macrophages. Surface IgM-positive, IgG-negative high-density B cells produced IgG antibodies for streptococcal carbohydrate in the presence of these T cells or tonsillar T cells. The culture supernatant solutions from these T cells or tonsillar T cells, however, failed to cause the B cells to produce IgG, indicating that class switching is not mediated by factors released from T cells. Lymphokines such as interleukin-2, human B cell growth factor, helper T cell factor, or interferon-gamma were also incapable of inducing IgG production. These results suggest that the cognate interaction between T cells and B cells is necessary for the immunoglobulin class switching.     

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.     

Leukocyte complement: interleukin-like properties of factor Bb

It has been previously shown that the activated form of Factor B (Factor Bb) of the alternative pathway of complement activation stimulates monocyte spreading and killing of xenogenic erythrocytes and staphylococci. Factor Bb also stimulates lymphocyte blastogenesis in vitro, and native (uncleaved) Factor B is a major constitutive product of murine macrophages. To evaluate the possible "monokine" or "lymphokine"-like properties of Factor Bb, a radioimmunoassay was developed to measure the quantities of Factor B in phytohemagglutinin (PHA)-mitogen-stimulated cultures of human peripheral blood mononuclear cells. Nonstimulated mononuclear cell cultures from human peripheral blood (containing 10-14% monocytes and greater than 85% lymphocytes) at a density of 3 X 10(6) cells/ml (in serum-free medium) released less than 7 X 10(-10) M/liter (60 ng/ml) of Factor B antigen in 24 hr at 37 degrees C, and when mononuclear cells were stimulated with PHA mitogen in serum-free medium, the levels of Factor B antigen in media at 24 hr were significantly higher 1-3 X 10(-8) M/liter (0.9-2.8 micrograms/ml). The molecular size of Factor B in these media was 50-65 kDa by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size appropriate for Factor Bb (60 kDa). Since pathological effects of macrophages in autoimmune disease may result from the release of lysosomal hydrolases, the effects of purified Factor Bb on mononuclear phagocytes were investigated in an in vitro system of murine peritoneal exudate macrophages. Factor Bb induced secretion of marker lysosomal hydrolases N-acetyl-beta-D-glucosaminidase (hexosaminidase) and beta-glucuronidase from thioglycollate-elicited murine peritoneal exudate macrophages in a dose-response and kinetic manner. Hydrolase release was induced in serum-free medium without a known particulate activator at a concentration of 80-200 nM (5-13 micrograms/ml) Factor Bb. Maximal release occurred in 3-5 hr at 37 degrees C and extracellular enzyme activity of hexosaminidase and glucuronidase increased as intracellular enzyme levels decreased, suggesting that Factor Bb triggers release of these enzymes from intracellular lysosomal pools. These results provide an example of a complement protein which is synthesized, released, and activated during mononuclear cell culture and which induces release of lysosomal enzymes from macrophages. In conventional terminology, Factor B or Factor Bb might be termed a "lymphokine," "monokine," or "interleukin".     

Human T4+ lymphocytes produce a phagocytosis-inducing factor (PIF) distinct from interferon-alpha and interferon-gamma

Unstimulated peripheral blood mononuclear cells from patients with angiocentric T cell immunoproliferative disorders and concanavalin A-stimulated normal peripheral blood mononuclear cells secrete a phagocytosis-inducing factor (PIF) that induces a fivefold to 50-fold enhancement of phagocytosis of IgG-coated ox red blood cells by U937 cells. We investigated the identity, production, and mechanism of the action of PIF. PIF activity was demonstrated in supernatants from nine of 44 phytohemagglutinin-stimulated interleukin 2 (IL 2)-dependent T cell lines and clones derived from purified T4+ cells, but was not found in supernatants from 26 lines and clones derived from phytohemagglutinin-stimulated T8+ cells. In addition, PIF was produced by four of four antigen-specific T cell lines and clones after stimulation with the appropriate antigen and antigen-presenting cells, and by HUT-102, a human T cell lymphotropic virus type I-transformed T cell line. PIF from all of these sources caused significant inhibition of U937 proliferation. This proliferation-inhibiting activity co-purified with phagocytosis-enhancing activity in sizing procedures and isoelectric focusing, which yielded an estimated m.w. of 35,000 to 55,000 and an estimated isoelectric point of 5.0 to 6.0 for PIF. In contrast, IL 2, recombinant interferon-alpha, and recombinant interferon-gamma had no effect on phagocytosis by U937 cells, and antibodies to interferon-alpha and interferon-gamma did not block the phagocytosis-inducing activity of PIF-containing supernatants. PIF appears to be a distinct lymphokine produced by a subset of T4+ lymphocytes, possibly those that proliferate in response to antigen. PIF may be important in the induction of erythrophagocytosis, which is associated with certain T cell immunoproliferative disorders.     

Comparison of in vitro tests for antioxidant and immunomodulatory capacities of compounds

Oxidative stress is considered to be critically involved in the normal aging process but also in the development and progression of various human pathologies like cardiovascular and neurodegenerative diseases, as well as of infections and malignant tumors. These pathological conditions involve an overwhelming production of reactive oxygen species (ROS), which are released as part of an anti-proliferative strategy during pro-inflammatory immune responses. Moreover, ROS themselves are autocrine forward regulators of the immune response.Most of the beneficial effects of antioxidants are considered to derive from their influence on the immune system. Due to their antioxidant and/or radical scavenging nature, phytochemicals, botanicals and herbal preparations can be of great importance to prevent oxidation processes and to counteract the activation of redox-regulated signaling pathways. Antioxidants can antagonize the activation of T-cells and macrophages during the immune response and this anti-inflammatory activity could be of utmost importance for the treatment of above-mentioned disorders and for the development of immunotolerance.Herein, we provide an overview of in vitro assays for the measurement of antioxidant and anti-inflammatory activities of plant-derived substances and extracts, by discussing possibilities and limitations of these methods. To determine the capacity of antioxidants, the oxygen radical absorbance capacity (ORAC) assay and the cell-based antioxidant activity (CAA) assay are widely applied. To examine the influence of compounds on the human immune response more closely, the model of mitogen stimulated human peripheral blood mononuclear (PBMC) cells can be applied, and the production of the inflammatory marker neopterin as well as the breakdown of the amino acid tryptophan in culture supernatants can be used as readout to indicate an immunomodulatory potential of the tested compound. These two biomarkers of immune system activation are robust and correlate with the course of cardiovascular, neurodegenerative and malignant tumor diseases, but also with the normal aging process, and they are strongly predictive. Thus, while the simpler ORAC and CAA assays provide insight into one peculiar chemical aspect, namely the neutralization of peroxyl radicals, the more complex PBMC assay is closer to the in vivo conditions as the assay comprehensively enlights several properties of immunomodulatory test compounds.