Lack of species specificity in the action of immunosuppressive factors of tumor cells and absence of competition between them and recombinant interleukin-2 and phytohemagglutinin for lymphocyte membrane receptors

We studied some possible mechanisms of action of immunosuppressor factors (ISF) produced by tumor cells on lymphocyte proliferation. ISF of murine tumor cell lines inhibited the mitogen induced proliferation of murine splenocytes as well as human mononuclear blood cells. Normal human mononuclear blood cells or concanavalin A-activated murine spleen cells preincubated with phytohemagglutinin (PHA) or interleukin 2 (IL-2) respectively, were strongly suppressed by ISF in response to these activators. When preincubated with splenocytes or blood cells for 2 h at 4 degrees C following washing, ISF suppressed the lymphocyte proliferation as effectively as when being with cells during all period of cultivation. ISF inhibited mitogen-induced lymphocyte proliferation at low dilutions. There was no competition for lymphocyte membrane receptors between these functionally heterogenic kinds of ISF. Collectively, these results show that ISF acted when being attached to some lymphocyte membrane receptors.     

Suppression of T lymphocyte chemotactic factor production by the opioid peptides beta-endorphin and met-enkephalin

The opioid peptides beta-endorphin and met-enkephalin have been shown to modulate human lymphocyte proliferation, mononuclear cell locomotion, natural killer cell activity, and neutrophil locomotion. This study demonstrates that beta-endorphin and met-enkephalin inhibit the production of a T lymphocyte chemotactic factor (LCF) by concanavalin A (Con A)-stimulated peripheral blood mononuclear cells. Inhibition of LCF production was observed by using concentrations of 10(-11) to 10(-6) M beta-endorphin or met-enkephalin but not alpha-endorphin. A bimodal pattern of suppression of LCF production was observed with both met-enkephalin and beta-endorphin when titrated from 10(-12) to 10(-6) M concentrations, with the peaks of suppressive activity occurring at concentrations of 10(-11) M and 10(-6) M. Timed studies of the production of LCF over a 54-hr period showed that there was an appreciable lag in the onset of measurable LCF activity in mononuclear supernatants produced in the presence of beta-endorphin and met-enkephalin. The suppression of LCF production mediated by opioid peptides in mononuclear supernatants was abrogated by depletion of glass-adherent mononuclear cells before culturing with opioids and Con A. The inhibitory effect of opioid peptides on LCF production was prevented by the addition of indomethacin to cell cultures. Additional experiments showed that exogenous prostaglandin E2 (PGE2) suppressed Con A-stimulated LCF production when added at concentrations ranging from 10(-6) to 10(-8) M. Other studies suggested that the mechanism of opioid peptide-mediated suppression of LCF production was due to an enhanced sensitivity of mononuclear cells to the inhibitory action of PGE2. These data provide further evidence for modulation of the immune response in humans by the neuroendocrine hormones beta-endorphin and met-enkephalin and further suggest a link between this modulation and arachidonic acid metabolism.     

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.     

Opioid peptides modulate production of interferon gamma by human mononuclear cells

The opioid neuropeptides have previously been shown to bind to and affect leukocyte function including lymphocyte proliferation, NK-cell activity, mononuclear cell chemotaxis, immunoglobulin synthesis, and lymphokine production. The effect of the opioid peptides beta-endorphin and Met-enkephalin on interferon gamma (IFN) production by concanavalin A-stimulated human mononuclear cells was examined. Both beta-endorphin and Met-enkephalin enhanced IFN production by the majority of donor mononuclear cells tested and did so at concentrations between 10(-14) and 10(-10) M. When 10(-12) M beta-endorphin or Met-enkephalin were included in concanavalin A-stimulated mononuclear cell cultures, IFN concentrations were significantly enhanced to 205 +/- 45 and 252 +/- 67% of control, respectively. Although the majority of cell preparations tested exhibited an enhanced production of IFN in response to these opioid peptides, some did not. When beta-endorphin or Met-enkephalin were utilized at 10(-11) M, 10 of 15 and 7 of 11 responded with IFN production greater than 20% above the control (untreated) level. There was not an absolute correlation between an enhanced response to beta-endorphin and Met-enkephalin, suggesting the presence of multiple receptor types on these cells for opioids. The opioid receptor antagonist, naloxone, did not significantly prevent the opiate effect. When 10(-8) M naloxone was included in cultures containing 10(-12) M beta-endorphin or Met-enkephalin no significant inhibition of the effect of either opioid on IFN production was observed.     

Functional properties of the 50 kd protein associated with the E-receptor on human T lymphocytes: suppression of IL 2 production by anti-p50 monoclonal antibodies

Monoclonal antibody 9.6 is specific for a 50 kd T cell surface protein (p50) associated with the sheep erythrocyte (E)-receptor on human T lymphocytes. This antibody interferes with many T cell functions. We have examined the effect of antibody 9.6 on lymphocyte proliferation and interleukin 2 (IL 2) production triggered by mitogens, soluble antigens, and alloantigens to elucidate the mechanism(s) of its immunosuppressive action. At concentrations as low as 50 ng/ml, 9.6 suppressed lymphocyte proliferation and the elaboration of IL 2 by T cells stimulated by PHA, alloantigens, or low concentrations of the phorbol ester TPA (less than or equal to ng/ml). Furthermore, in cultures stimulated by a combination of PHA plus TPA, 9.6 did not inhibit the acquisition of IL 2 receptors but inhibited proliferation and IL 2 production. Immunoaffinity-purified IL 2 completely restored lymphocyte proliferation in cultures inhibited by 9.6. Studies of kinetics of inhibition by 9.6 showed that this antibody inhibited lymphocyte proliferation induced by PHA, alloantigen, and PPD even when added at 24, 48, and 72 hr, respectively, after the initiation of these cultures, suggesting that 9.6 does not block lectin binding or antigen recognition by T cells and that it can inhibit lymphocyte proliferation even after cells have undergone one or more rounds of cell division. A dose-response analysis of lymphocyte proliferation induced by PHA or by TPA demonstrated that the degree of inhibition by 9.6 decreased with increasing concentrations of these mitogens. Antibody 9.6 did not inhibit lymphocyte response induced by optimal concentrations of PHA (50 to 100 micrograms/ml; PHA-M) but inhibited proliferation of maximally induced lymphocytes by using a synergistic combination of low concentrations of PHA (5 micrograms/ml, PHA-M) plus TPA (1 ng/ml). Taken together, these findings indicate that 1) 9.6 inhibits lymphocyte proliferation by affecting IL 2 production, 2) 9.6 does not inhibit the acquisition of 9.6 receptors induced by a synergistic combination of PHA plus TPA, and 3) p50 molecules may be involved in multiple pathways of T cell activation.     

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".     

Effect of cholecystokinin and gastrin on human peripheral blood lymphocyte functions, implication of cyclic AMP and interleukin 2

The effects in vitro of sulphated and desulphated cholecystokinin (CCK)-8, and of gastrin-17 and gastrin-34 were studied at concentrations from 10⁻¹⁴ M to 10⁻⁶ M on several functions of human peripheral blood lymphocytes, i.e.: adherence to substrate, mobility (spontaneous and directed by a chemical gradient or chemotaxis), and spontaneous and phytohaemagglutinin (PHA)-mediated proliferation. All peptides, at concentrations from 10⁻¹⁰ M to 10⁻⁸ M, inhibited significantly the mobility capacity and PHA-induced proliferation, and increased the adherence and spontaneous proliferation. A dose-response relationship was observed, with a maximum response of lymphocyte functions at 10⁻¹⁰ M. These peptides induced a significant increase of intracellular cAMP levels at 30 and 60 sec. Because lymphoproliferation requires production of interleukin 2 (IL-2) by lymphocytes, we also measured the IL-2 production in the presence of the CCK and gastrin peptides, finding that this production was higher than in the respective controls. When peptides were added to samples containing PHA, the IL-2 production was significantly decreased with respect to samples incubated with PHA alone. These results suggest that the CCK and gastrin peptides are negative modulators of lymphocyte mobility (spontaneous mobility and chemotaxis), causing an inhibition of these activities through an increase of intracellular cAMP levels, and of PHA-induced lymphoproliferation, which is mediated by a diminution of the IL-2 production by lymphocytes.     

Biological activities of interleukin-2 and gamma-interferon in bovine T lymphocyte conditioned media

Interleukin 2 and gamma-interferon were revealed in cultures of Concanavalin A-activated bovine mononuclear cells from peripheral blood leukocytes and their kinetics of production were described. Cyclosporin A could dramatically inhibit the synthesis of interleukin 2, whereas it did not affect the interleukin 2-dependent proliferation of bovine T blasts. Bovine T lymphocyte conditioned media (TLCM) containing gamma-interferon increased the expression of beta-2 microglobulin in human HEL (Human Embryo Lung) 299 cells and exerted a potent anti-proliferative effect upon bovine Aubek cells. The beta-2 microglobulin modulating activity of bovine gamma-interferon was exerted at very low concentrations, which showed no detectable antiviral activity on human cells.     

Biochemical characterization of lymphocyte regulatory molecules. II. Purification of a class of rat and human lymphokines

Rat spleen cells activated in vitro by concanavalin A produce lymphokine molecules that possess biologic activity in a number of murine lymphocyte response assays. A single class of lymphokine most adequately described as T cell growth factor (TCGF, Interleukin-2) with a m.w. of 15,000 as estimated from gel filtration studies and with an isoelectric range of 5.4 to 5.6 stimulates i) the growth of established T cell lines in culture, ii) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is non-mitogenic, iii) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) mouse spleen cell cultures, and iv) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures and in nude mouse spleen cell cultures. We suggest that in each of the assay systems tested, this class of rat lymphokine acts directly on activated T cells. Nonactivated T cells must be stimulated by either mitogen or antigen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued lymphokine-dependent proliferation. Similarly, human peripheral blood mononuclear cells activated by phytohemagglutinin (PHA) produce a class of lymphokines of identical size with an isoelectric point of 6.0 to 6.5 that possess the same biologic properties as measured in murine lymphocyte response systems.     

B-cell-derived interleukin 1 (IL-1)-like factor. I. Relationship of production of IL-1-like factor to accessory cell function of Epstein-Barr virus-transformed human B-lymphoblast lines

The relationship of production of interleukin 1 (IL-1)-like factor to accessory function of Epstein-Barr virus (EBV)-transformed B lymphocytes was examined. Six of eight human EBV-B cell lines spontaneously produced and released detectable levels of thymocyte comitogenic factor in vitro, but no interleukin 2 (IL-2) activity. Eight of eight produced fibroblast proliferation activity. Culture supernatants from the two apparent nonproducers of thymocyte comitogenic activity induced the proliferation of the IL-1-dependent murine helper-T-cell clone D10G4.1 in the presence of concanavalin A (Con A). One of the EBV-B cell lines produced a potent inhibitory factor in addition to IL-1-like thymocyte comitogenic and fibroblast proliferation factors. The inhibitory factor inhibited mouse thymocyte proliferative response to Con A, and the proliferation of the IL-2-dependent CT6 cell line, but not human fibroblast growth. All but one of the eight EBV-B cell lines tested, the exception being the line that produced an inhibitory factor, were able to serve as antigen-presenting cells that enabled purified human T lymphocytes to proliferate in one-way mixed lymphocyte reactions (MLR) and in response to Con A. The supernatants of 14 of 16 clones derived from two of the EBV-B cell line cells contained thymocyte comitogenic activity and all 16 stimulated fibroblast proliferation. The phenotypic characteristics of the EBV-B cell lines were heterogeneous, but there was no clear-cut relationship between the cell surface phenotypes of either the cloned or uncloned EBV-B cells and their ability to produce these factors. These studies show that all of the EBV-B cell lines that can function as accessory cells have the capacity to produce an IL-1-like factor.     

Calcium ionophore (A23187) increases interleukin 1 (IL-1) production by human peripheral blood monocytes and interacts synergistically with IL-1 to augment concanavalin A stimulated thymocyte proliferation

The effects of calcium ionophore, A23187, on production of interleukin 1 (IL-1) by human peripheral blood monocytes (PEMo) and on murine thymocyte proliferation were examined. A23187 induced IL-1 production by human PBMo. The optimal dose was 10(-6) M. Although IL-1 production induced by A23187 was less than that by lipopolysaccharide (LPS) or silica, A23187 together with LPS had a synergistic effect on induction of IL-1. A23187 also had a more marked synergistic effect in concert with Concanavalin A and/or IL-1 on murine thymocyte proliferation. The optimal dose was also 10(-6) M. This represents the first report suggesting that monocytes or the monocyte product, IL-1, may contribute to the mitogenic effect of A23187 for thymocytes.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Immunomodulating activity in supernatants from EBV immortalized lymphocytes

Summary Our earlier work has demonstrated that EBV immortalized B lymphocytes are involved in a factor dependent autostimulatory cycle. Soluble growth stimulating activity was released into culture supernatants by these growing B cells. Growth enhancing (GE) media from B lymphocyte lines, immortalized by EBV infection, contained soluble factor(s) which modulated the Con A response of normal human mononuclear cells. Conditioned media from these lines affected the Con A response in a biphasic manner, stimulating the blastogenic response at lower concentrations, while inhibiting at higher concentrations. At stimulatory concentrations, the blastogenic response to Con A began earlier than in controls and was markedly enhanced by day 2. GE media reduced the initial response of purified B cells to pokeweed mitogen. GE media did not support growth of IL-2 dependent cells. GE media from some EBV-carrying B cell lines had measurable IL-1 activity in the mouse thymocyte PHA response. GE media from LPS stimulated B lymphocyte lines produced significant IL-1-like effects on stimulated mouse thymocytes. These results suggested that these B cell lines may produce IL-1-like factors that cooperate in T cell responses. The possibility that such factors may play a role in B lymphocyte transformation by EBV is discussed.Supported by grants from the Concern Foundation and the Brigham Surgical Foundation     

IL-12, IL-6 and IFN-gamma production by lymphocytes of pregnant women with rheumatoid arthritis remission during pregnancy

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease with progressive activity. The RA remission was observed in women during pregnancy, but the mechanism responsible for remission is hypothetical only and concerns mechanisms of immune regulation such as lymphocyte subpopulations and interleukin production. AIMS: The lymphocyte subpopulations and interleukin production in vitro in a group of healthy non-pregnant women, healthy pregnant women and pregnant women suffering from RA may help towards a better understanding of regulation of the immune processes. METHODS: The investigations were performed in trimester III--2 days after delivery and 6 weeks after delivery. Peripheral blood lymphocytes were isolated on Gradisol gradient and analysed immediately or after having been cultured for 72 hours in RPMI medium supplemented with 10% FCS. The cultures were terminated after 72 h, supernatants stored at -72 degrees C for interleukin evaluation. The concentrations of IFN-gamma, IL-2, IL-6, IL-12, TNF-alpha and its soluble receptors R-I, R-II were estimated in non-stimulated and PHA (Sigma, 5 microg/ml) stimulated culture supernatants using ELISA Endogen kits according to the manufacturer''s instructions. RESULTS: The general pattern of T cell subpopulation distribution was similar in all analysed groups. Decreased IFN-gamma, IL-12 and increased IL-6 production by lymphocytes after PHA stimulation was found in trimester III in pregnant women with RA as compared to healthy pregnant woman. CONCLUSION: The obtained results suggest that in pregnant women with RA the TH1 cell response predominates, contrary to healthy pregnant women with TH2 type functional response. These phenomena were not observed after delivery.     

Leukotrienes augment interleukin 1 production by human monocytes

The effects of leukotrienes (LT) on production of interleukin 1 (IL 1) by human peripheral blood monocytes were examined. LTB4 enhanced IL 1 production by lipopolysaccharide (LPS)-stimulated monocytes twofold to threefold, and the most efficient concentrations of LTB4 were 10(-8) to 10(-7) M. LTD4 also enhanced IL 1 production, but to a lesser extent than LTB4. Adherence-purified, but otherwise unstimulated, human monocytes could also be induced to produce IL 1 in response to LTB4. Similarly, IL 1 production by monocytes stimulated with the known IL 1 inducers muramyl dipeptide, silica, or zymosan was also enhanced by LTB4. Inhibition of cyclooxygenase with use of indomethacin during IL 1 production by LPS-treated monocytes enhanced thymocyte response to IL 1, but LTB4 further enhanced IL 1 production when added to indomethacin-treated monocyte cultures. Neither LTB4 nor indomethacin had any direct effect on thymocyte proliferation. Optimal enhancement of IL 1 production occurred when LPS and LTB4 were present together at the initiation of the 24-hr monocyte culture. Significant enhancement was also observed, however, when monocyte cultures were either preincubated with LTB4 before addition of LPS or cultured with LPS alone for 3 hr before addition of LTB4. These results indicate that leukotrienes can modulate IL 1 production by human monocytes and suggest that they may play a role in IL 1-mediated functions of monocytes in inflammatory and immune reactions.     

Monoclonal antibodies to CD2 and lymphocyte function-associated antigen 3 inhibit human thymic epithelial cell-dependent mature thymocyte activation

Recent study of human thymocyte-thymic epithelial (TE) cell interactions has demonstrated that thymocytes bind to TE cells, and a consequence of this binding is the provision of accessory cell signals by TE cells for phytohemagglutinin (PHA)-induced mature thymocyte activation. In this paper we report on studies of the molecules involved in TE cell-dependent mature thymocyte activation. TE-thymocyte interactions necessary for PHA-induced thymocyte activation were inhibited by monoclonal antibodies against the cluster of differentiation (CD)2 antigen on thymocytes and lymphocyte function-associated (LFA)-3 antigen on TE cells. Inhibition of TE accessory cell signals by antibodies against CD2 (alpha CD2) and LFA-3 (alpha LFA-3) antigens occurred early on during thymocyte activation and prevented thymocyte interleukin 2 receptor expression. Further, alpha CD2 and alpha LFA-3 inhibited PHA-induced thymocyte activation in whole thymic explant cultures suggesting a significant role of the CD2 and LFA-3 antigens in thymocyte activation when accessory cell signals for PHA-induced thymocyte triggering were delivered by cells within an intact thymic microenvironment.     

Ability of human leukocytic pyrogen to enhance phytohemagglutinin induced murine thymocyte proliferation

Human leukocytic pyrogen, a monokine produced by stimulated human mononuclear phagocytes, will enhance the murine thymocyte proliferation response to phytohemagglutinin (lymphocyte activating factor (LAF) activity). During all steps of purification of human LP, pyrogenicity and LAF activity are coincidental suggesting a single identity for the two monokines. The LAF assay for human LP is highly sensitive and can detect human LP at a concentration of 10^?12M. Further experiments suggests that human LP and LAF activities could be destroyed by heating to 70 °C. Furthermore, while in vivo pyrogenicity of human LP can be blocked by ibuprofen, the in vitro LAF activity of the same molecule is unaffected by ibuprofen. Immune rabbit serum directed against human LP could also block in vitro LAF activity either by preincubation with LP or by blocking during culture.     

Effect of catalase on the proliferation of human lymphocytes to phorbol myristate acetate

Phorbol esters have been documented to stimulate the proliferation of human blood mononuclear cell cultures. In addition, these agents are also known to stimulate the production and release of reactive oxygen species by monocytes. We demonstrated previously that H2O2, one of these oxygen metabolites, impairs the proliferative capacity of human blood lymphocytes. Therefore, in these experiments, we determined whether or not the H2O2 released by monocytes after activation by PMA modifies the proliferation of lymphocytes to this agent. Human blood mononuclear cells (80% lymphocytes and 20% monocytes) were incubated with PMA, and lymphoblastic transformation (LBT) was quantitated at 3 and 5 days by pulsing the cultures with thymidine. Initial experiments established that the concentration of PMA required for optimal LBT was 50 ng/ml. We then demonstrated that this concentration of PMA also induces a burst in hexose monophosphate shunt activity and H2O2 production of mononuclear cells as indicated by the enhanced oxidation of 14C-glucose and 14C-formate, respectively. The amount of H2O2 released into the medium was substantial. Our measurements indicate that the concentration of H2O2 could reach values as high as 0.008 mM during the first 2 hr of the cultures. The addition of catalase to PMA-treated cultures in concentrations sufficient to scavenge the H2O2 released by the monocytes was associated with an enhanced thymidine uptake (mean 79%). These results indicate that the hydrogen peroxide released by the monocytes modifies the response of lymphocytes to the PMA. Paradoxically, mononuclear cell cultures depleted of monocytes also had a lower proliferation to PMA than mononuclear cell cultures. This observation indicates that monocytes also produce factors required for lymphocyte proliferation to PMA such as an interleukin. In contrast, to PMA cultures, catalase did not alter the proliferation of mononuclear cell cultures stimulated by PHA. We previously documented that PHA does not stimulate an immediate burst in the oxidative metabolism of mononuclear cultures. Therefore, the effect of catalase in these two culture systems appears to correlate with the capacity of the mitogen to stimulate the oxidative metabolism of mononuclear cells. These observations suggest that the release of reactive oxygen species by monocytes may modify the response of lymphocytes to antigens both in vitro and in vivo.     

Alpha melanocyte stimulating hormone inhibits immunostimulatory and inflammatory actions of interleukin 1

The ability of interleukin 1 (IL 1) to augment the proliferation of murine thymocytes in vitro was inhibited in a dose-dependent manner by the neuropeptide alpha-melanocyte-stimulating hormone (alpha MSH). The minimal effective concentration of alpha MSH was 10(-11) M. Maximal effect occurred between 10(-8) and 10(-7) M, with diminishing effectiveness at higher concentrations. IL 1-induced production of prostaglandin E (PGE) by fibroblasts was also inhibited by alpha MSH with a biphasic dose response. The minimal effective concentration was 10(-11) M, and maximum effect was achieved at 10(-10) M. alpha MSH appeared to affect the interaction of IL 1 with its target cells in a specific manner, because it did not inhibit basal mitogen-induced thymocyte proliferation or IL 2-induced proliferation of a cytotoxic T lymphocyte line. Furthermore, production of IL 1 by endotoxin-stimulated monocytes was not affected by alpha MSH. An analog of alpha MSH (Nle4, D-Phe7 alpha MSH), which is highly potent in other melanotropin-sensitive systems, did not affect the action of IL 1 on thymocytes, suggesting that the immunomodulatory effects of alpha MSH may not be mediated by the classic melanocyte alpha MSH receptor. The influence of alpha MSH on thymocytes and fibroblasts suggests that alpha MSH is an endogenous antagonist of IL 1, perhaps important for limiting inflammatory damage to host tissues.     

Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus

Wheelock, Frederick E. (Western Reserve University, Cleveland, Ohio). Virus replication and high-titered interferon production in human leukocyte cultures inoculated with Newcastle disease virus. J. Bacteriol. 92:1415-1421. 1966.-High titers of interferon (20,480 culture-protecting units per ml) are produced in freshly prepared human leukocyte cultures inoculated with a Newcastle disease virus (NDV)-cell multiplicity of 1:1. NDV replicates to low titers in these cultures. Incubation of leukocytes at 37 C for 24 hr prior to inoculation of NDV results in almost complete loss of detectable interferon production, but virus replicates to higher titers than in the freshly prepared cultures. In contrast, no diminution of interferon production in response to phytohemagglutinin (PHA) occurs on 24 hr of incubation of cultures prior to addition of PHA. Experiments with cultures of predominantly pure cell fractions of peripheral blood indicate that the lymphocyte fraction produces interferon in response to either NDV or PHA, and that polymorphonuclear leukocytes produce no interferon in response to these agents. These studies suggest a hitherto unsuspected ability of human lymphocytes to produce high titers of interferon in vivo.