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Activated antigen-presenting cells and central nervous system microglia produce IL-1 beta, TNF-alpha, IL-6, and PGE-1. These monokines participate in the lymphocyte activation, demyelination, and intrathecal immunoglobulin synthesis seen in multiple sclerosis (MS). Exacerbations of MS are ameliorated by IFN-beta, but provoked by IFN-gamma, possibly through an effect on monocytes (Mo). We studied the effects of IFNs and PG on monokine secretion under stringent low-endotoxin conditions. Spontaneous and IFN-gamma-induced IL-1 beta secretion was greater in MS than in NL Mo. IFN-beta did not inhibit IFN-gamma-induced secretion of monokines, which contrasts with IFN-beta's inhibitory effect on IFN-gamma-induced MHC class II expression. PGE1, a cAMP agonist, caused a 30-fold induction of IL-6 secretion. Indomethacin directly inhibited this induction. Low-dose IFN-beta, through effects on T cells, and cAMP agonists, through effects in T cells and Mo, may ameliorate inflammatory diseases characterized by excessive monokine secretion.

Monokines IL12, IL15, and IL18 have been shown to activate NK cell function, however with high apoptosis induced by their combination within 48 h. Here, we demonstrate for the first time that CD56+ cells incubated for only 18 h with the combination of IL15/IL12 or IL15/IL18, then washed, and further cultured in plain medium, exhibit low levels of apoptosis. These shortly activated CD56+ cells show high killer activity against NK- and LAK-sensitive tumor targets that persists over a culture period of 18 days after two additional 6 h cycles of exposure to the monokines applied every 8 days and also retain their ability for high cytokine production during each exposure. Moreover, these repetitive short-term exposures of CD56+ cells to the monokine combinations result in long-lived CD56+ cells with slower rates of FcgammaRIII receptor (CD16) decline, therefore exhibiting higher antibody depended cytotoxicity, as opposed to the continuous incubation with the monokine combinations. In conclusion, short-term exposure of CD56+ cells to IL15/IL12 or IL15/IL18 at 8-day intervals may hold a promise for improved clinical results in cellular adoptive cancer immunotherapy and for the in vivo injections of the monokines.

Human placental protein 14 (PP14, also referred to as glycodelin and progesterone-associated endometrial protein) inhibits phytohemagglutinin (PHA)-stimulated T-cell proliferation and monokine secretion within PBMC populations. However, the mechanisms underlying these and other PP14 immunoinhibitory activities remain unclear. In the present study, we asked whether PP14's T-cell inhibitory effect is a direct one or, alternatively, an indirect consequence of accessory cell (AC) perturbation. Using either immunopurified PP14 or first-trimester amniotic fluid (AF) as a rich source of PP14, we documented inhibition of the proliferation of highly purified peripheral blood T-cells when stimulated with anti-CD3 mAbs or PHA in the presence of paraformaldehyde-fixed AC. Significantly, PP14 inhibited T-cell proliferation and IL-2 secretion induced by immobilized anti-CD3 and anti-CD28 mAbs in the absence of AC. PP14 depletion (via immunoprecipitation) abrogated AF's T-cell inhibitory activity, indicating that the PP14 within the amniotic fluid is required for this immunoregulatory effect. These findings establish that PP14 can inhibit T-cell proliferation in the absence of AC and thus add PP14 to the relatively restricted set of immunoinhibitory proteins that are known to target T-cells directly. Additional data demonstrate that PP14's inhibitory effect can be overridden by stimuli which circumvent early events during T-cell receptor (TCR) activation, namely, protein kinase C activators in combination with Ca2+ ionophores. These latter results suggest that PP14 inhibits early events in the TCR signaling pathway.

Interleukin 12 (IL-12) is a monokine which plays a critical role in resistance to Mycobacterium tuberculosis infection. However, little is known about the regulation of IL-12 production by human cells stimulated with BCG. Here we report that in vitro infection of human mononuclear cells with M. bovis BCG induces the release of IL-12 protein. We also observed that the ability of BCG to stimulate release of IL-12 from human cells was significantly inhibited by IL-10. The inhibitory effect of IL-10 on the secretion of IL-12 was specific, as it was significantly abolished in the presence of anti-IL-10 neutralizing monoclonal antibody. These results were further confirmed as anti-IL-10 antibodies markedly increased the levels of IL-12, suggesting that BCG-induced IL-10, as well as exogenous IL-10, can regulate IL-12 production by human cells stimulated with M. bovis BCG. Interestingly, IFN-gamma production in response to BCG had no significant increase by the addition of neutralizing antibodies to IL-10. Moreover, anti-IL-12 antibodies markedly reduced the levels of IFN-gamma produced by BCG-stimulated human cells and abrogated the capacity of anti-IL-10 to increase BCG-induced IFN-gamma. These studies are the first to demonstrate a regulatory effect on IL-12 production by human cells infected with M. bovis BCG and at the same time suggest that IL-12 may play an essential role during the human immune response to M. bovis BCG stimulation.

Elevated levels of circulating monokines (IL-6, IL-1, and TNFα) have been seen in HIV-1 infection, and the overproduction of these cytokines could contribute to AIDS pathogenesis in various ways. In previous work, we had seen that exposure of human monocytes to HIV-1, including inactivated, noninfectious HIV, led to rapid IL-6 gene expression and secretion. To investigate cytokine production in response to components of HIV by monocytes/macrophages, production of IL-6 and IL-10 were examined in a human monocytic cell line, THP-1, stimulated by HIV proteins. IL-6 production was induced in THP-1 cells by a detergent lysate of HIV, particularly fractions at molecular weight of 25-50 kDa. Recombinant HIV envelope glycoprotein 41 (gp41), but not gp120 or p24, also was seen to induce significant IL-6 production by THP-1 cells. These results suggest that gp41, transmembrane protein, is the primary HIV-encoded protein involved in inducing IL-6 production. IL-10 was also produced with delayed kinetics, following IL-6 production in THP-1 cells stimulated by gp41. To investigate a possible regulatory role for IL-10 in HIV-induced monokine production, recombinant IL-10 was added to gp41-exposed THP-1 cells. IL-10 inhibited gp41-induced IL-6 production and reduced the expression of IL-6 mRNA. When anti-human IL-10 neutralizing antibody was added to THP-1 cells, IL-6 production was enhanced. These results suggest that the IL-6 production may be downregulated by endogenously produced IL-10 and that IL-10 may downregulate cytokine production by HIV-activated monocytes via an autoregulatory mechanism.

Dietary exposure to n-3 fats found in marine fish oils are known to reduce certain inflammatory conditions. Although depressed prostaglandin E2 (PGE2) production is thought to be a major mechanism of the beneficial effects, the direct effects of n-3 fatty acids on inflammatory macrophage function are not well understood. In this study, production of the inflammatory monokine, tumor necrosis factor-alpha (TNF alpha), by isolated murine macrophages was assessed following a 3-week feeding with diets containing either 10% menhaden fish oil as a source of n-3 fatty acids or, as a control and source of n-6 fatty acids, 10% safflower oil. Cultures of peritoneal macrophages from mice fed diets with n-3 fatty acids had more TNF alpha activity 24 hr after in vitro stimulation with bacterial lipopolysaccharide than did macrophages from mice fed the n-6-containing diet. The onset and maximal synthesis of bioactive TNF alpha and down-regulation of messenger RNA for TNF alpha appeared to be similar for the two diets, suggesting that macrophages from mice fed a diet high in n-6 but not n-3 fatty acids were capable of removing active TNF alpha from culture media. Experiments in which PGE2 was added exogenously indicated that the removal of TNF alpha from culture supernatant by macrophages was induced by lower concentrations of PGE2 than that associated with termination of production, and that n-3 fatty acid diets caused a selective loss in the clearance mechanism. These results demonstrate a specific alteration of PGE2-mediated regulation of macrophage-produced TNF alpha by n-3 fatty acids.

Zinc is an important trace element for immune function. The mechanisms by which zinc ions interact with immune cells are, however, still poorly understood. In the present study, we succeeded in defining transferrin and insulin as proteins which selectively enhance zinc-induced monokine induction in peripheral blood mononuclear cells (PBMC). An involvement of the transferrin receptor and the insulin receptor was ruled out. Zinc stimulation of PBMC resulted in an increase of intracellular free zinc, measured by a zinc-specific fluorescence probe, zinquin, the amount of which could be raised by substitution of neither transferrin nor insulin. Inhibition of second messengers by herbimycin A and HA 1004 revealed a participation of protein tyrosine kinases and of cAMP- and cGMP-dependent protein kinases in zinc-induced monokine secretion. We therefore suggest that zinc acts synergistically with stimulants of the above-mentioned signal transduction pathways by direct influence on the second messenger niveau.

Recent evidence has demonstrated the importance of substance P and its receptor in macrophage-mediated inflammatory responses. While previous studies have shown that substance P can augment proinflammatory monokine production, little is known about the effects of this neuropeptide on the production of monokines that might limit inflammation. In the present study we have investigated the effect of substance P treatment on the production of transforming growth factor-beta 1 (TGF-beta 1) in cultured murine macrophages. We report that, while substance P agonist alone elicited increases in TGF-beta 1 mRNA expression and modest increases in TGF-beta 1 secretion, substance P dramatically diminished LPS- or IFN-gamma-induced TGF-beta 1 production. These results suggest a previously unrecognized mechanism where substance P may act as a proinflammatory mediator by limiting the production of excessive levels of TGF-beta 1 by LPS- or IFN-gamma-activated macrophages.

We have shown that continuous treatment of mice from birth to adulthood with neutralizing anti-IL-10 antibodies leads to specific depletion of Ly1 B cells, while conventional B cells remain normal in terms of number, phenotype, and function. Extending our characterization of these animals, we show here that anti-IL-10 treated mice can be distinguished from untreated or isotype control treated mice by several other criteria. Anti-IL-10 treated mice contained substantially elevated levels of circulating TNF-alpha, and in many cases circulating IL-6, and were profoundly susceptible to death by LPS-induced shock, a monokine mediated inflammatory reaction. Analysis of serum immunoglobulin levels in anti-IL-10 treated mice revealed a decrease in serum IgA levels to accompany the previously reported reduction in serum IgM, plus a striking increase in IgG2a and IgG2b levels. Further investigation of the Ly1 B cell depletion of anti-IL-10 treated mice revealed that this effect was transient as evidenced by the return of Ly1 B cells in normal numbers 8 weeks after anti-IL-10 treatment was discontinued. The Ly1 B cell depletion that occurred during anti-IL-10 treatment was found to be compensated by an increase in peritoneal T cells and granulocytes. Finally, while anti-IL-10 treated mice were unable to produce antibodies to phosphorylcholine and alpha 1,3-dextran, they developed normal antibody responses following intraperitoneal injections of TNP-Ficoll, suggesting the existence of subcategories within the family of thymus independent type II polysaccharide antigens. These data are discussed within the context of their implications for the roles of IL-10 and Ly1 B cells in the immune system.

Interleukin-1 is a potent stimulator of arachidonic acid (AA) metabolism and this activity could be attributed to the activation of the prostaglandin-forming enzyme cyclooxygenase or of the arachidonic-releasing enzyme phospholipase A2 or both. Prostaglandin E2 (PGE2), a cyclooxygenase product, and LTB4 (5-(S),12-(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a lipoxygenase product, are potent mediators of inflammation. Recently a new cytokine produced by macrophages and named interleukin-1 receptor antagonist (IL-1ra) (MW 22,000 Da) which specifically binds and blocks IL-1 receptors, has proven to be a potent inflammatory inhibitor. In our studies we found that monocyte suspensions, pretreated with hrIL-1ra at increasing concentrations (0.25-250 ng/ml) for 10 min and then treated with LPS in an overnight incubation inhibits, in a dose-dependent manner, the generation of LTB4 as measured by the highly sensitive radioimmunoassay method. In monocytes pretreated with hrIL-1ra (250 ng/ml) for 10 min and treated with arachidonic acid (10(-5)-10(-9) M) and LPS overnight, the release of LTB4 was partially inhibited when compared to hrIL-1ra-untreated cells. Moreover, hrIL-1ra (250 ng/ml) caused a partial inhibition of monocyte LTB4 production when the cells were activated with AA (10(-7) M) and then treated with IL-1 beta (5 ng/ml) overnight or 24 hr incubation. In addition, human monocytes pretreated for 10 min with increasing doses of hrIL-1ra (0.25-250 ng/ml) and then treated with hrIL-1 alpha (5 ng/ml) or beta (5 ng/ml) for 18 hr, also resulted in the inhibition of PGE2 generation as measured by RIA when compared with hrIL-1ra-untreated cells. When the cells were treated with hrIL-1ra (250 ng/ml) and activated for 18 and 48 hr with increasing doses of hrIL-1 beta a strong inhibitory effect was found on PGE2 production. HrIL-1ra used at 15 ng/ml gave a partial inhibition of LTB4 generation, after LPS (1-100 ng/ml) treatment, while NDGA totally blocked the production of LTB4. Moreover, PGE2 released by macrophages activated with LPS (100 ng/ml) or hrIL-1 beta (5 ng/ml) at 18 hr incubation time was strongly inhibited when hrIL-1ra (250 ng/ml) was used. These data suggest that the inhibition of LTB4 and PGE2 by this new macrophage-derived monokine IL-1ra occurs through the block of the IL-1 receptor, rather than phospholipase A2, and thus IL-1ra may offer a potential therapeutic approach to inflammatory states.

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".

T-cell-activating monokines of guinea pigs with interleukin 1-like activity were produced in protein-free medium by stimulation of peritoneal macrophages with synthetic muramyl dipeptide (MDP). Gel filtration of the culture supernatant of MDP-stimulated macrophages revealed that most of the activity to potentiate the responses of thymocytes and lymph node T cells to phytohemagglutinin were found in the high-molecular-weight (40,000-80,000) fraction. By isoelectric focusing, the monokine in the high-molecular-weight fraction was focused at a pI of around 4.7 forming a somewhat broad band, indicating some heterogeneity. In the analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the high-molecular-weight fraction, the peak of the activity was found at an approximate Mr of 60K under unreduced as well as reduced conditions. Accordingly, it is likely that the monokine in this fraction is a polypeptide of 60K, but a possibility still remains that the 60K component is an aggregate of low-molecular-weight monokine which requires more rigorous conditions for dissociation.

The restorative effect of thymosin fraction 5 (TF5) on the thymus of γ-irradiated mice was examined. Four different mouse strains were used in this study since earlier work determined that the degree of response to TF5 is strain dependent. The responsiveness to comitogenic effect of interleukin 1 (IL-1) was used to measure the rate of recovery of immunocompetent cells in the thymus, since only more mature PNA − , Lyt-1 + -2 − medullary cells respond to this monokine. Contrary to several earlier reports that radioresistant cells repopulating the thymus within the first 10 days after irradiation are mature, corticosteroid resistant, immunocompetent cells, the thymic cells from irradiated mice in all strains used had greatly reduced responses to IL-1. Daily intraperitoneal injections of TF5 increased significantly the responses of thymic cells to IL-1 in 10- to 13-weeks-old C57Bl/KsJ, C57B1/6, C3H/HeJ, and DBA/1 mice. Older mice, 5 months or more in age, of DBA/1 strain did not respond to treatment with TF5. However, C3H/HeJ mice of the same age were highly responsive. In conclusion, (1) cells repopulating the thymus within 12 days after irradiation contain lower than normal fraction of mature IL-1 responsive cells, (2) thymic hormones increase the rate of recovery of immunocompetent cells in the thymus, and (3) the effect of thymic hormones is strain and age dependent.

Lymphocyte subsets separated on the basis of nylon-wool adherence and E and EA rosetting, and characterized for the presence of esterase-positive phagocytic cells were investigated for production of leukocyte migration inhibitory factor (LIF) in response to polyclonal T- and B-cell activators, PHA, ConA, PWM, and Epstein-Barr virus (EBV). In the nylon-passed population only the high avidity E+EA+ cells responded to ConA, PHA-induced LIF production in all E-rosetting subsets. The nylon-adherent E+ subset, which contains activated T cells, produced LIF spontaneously. B cells produced LIF when exposed to PWM or uv-inactivated EBV. In accordance with the known T-cell dependence of PWM activation, LIF was detected only in supernatants of reconstituted populations containing both B and T cells. In contrast, uv-inactivated EBV, devoid of transforming potential, elicited LIF production in the pure B-cell population. LIF production in response to polyclonal activators seemed to be independent of accessory cells since reconstitution with autologous macrophages or semipurified monokine, high-molecular-weight Interleukin 1 (IL-1), did not alter the results.

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 −12 M . 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.

We have investigated the relationship between the monokine interleukin 1 (IL-1) and the connective tissue-stimulating activities produced by monocytes such as mononuclear cell factor (MCF). Using almost exclusively human tissue we have monitored a wide range of MCF-like activities through the partial purification of IL-1 by gel filtration and isoelectric focusing. Activities measured include stimulation of chondrocytes to produce prostaglandins, plasminogen activator and proteoglycanase, enhancement of synovial cell proliferation, and stimulation of cartilage resorption, in addition to IL-1 (lymphocyte activating factor) activity. The activities described show the same molecular heterogeneity; the active material has similar potencies in the different systems, and removal of IL-1 activity by pretreatment with phenylglyoxal also results in loss of the connective tissue-stimulating activities. These results show that the factors responsible for this wide range of activities are very closely related to IL-1 and give further evidence in support of the possible involvement of IL-1 in the processes of joint destruction occurring in chronic inflammatory conditions such as rheumatoid arthritis.

An increase in the concentration of the acute-phase reactant, serum amyloid A (SAA), following endotoxin treatment, is a consequence of the action of lipopolysaccharide (LPS) on macrophages to produce a monokine, the SAA inducer, which in turn, triggers SAA synthesis by hepatocytes. We have found that murine SAA inducer is closely related, if not identical, to murine lymphocyte activating factor (LAF), otherwise known as Interleukin 1 (IL 1). Furthermore, both rabbit endogenous pryrogen (EP), which is believed to be identical to LAF (IL 1), and human LAF (IL 1), induced elevated SAA concentrations in C3H/HeJ mice. Antiserum previously shown to block both pyrogenic and thymocyte proliferating activities of the species of rabbit EP exhibiting an isoelectric point of pH 7.3 (EP 7), also blocked the SAA inducing activity of EP7. Phenylglyoxal treatment of highly purified murine LAF (IL 1) abrogated both thymocyte proliferating activity and the SAA inducing activity. These studies support and extend previous reports suggesting that within 2 hr of an inflammatory stimulus, macrophages produce a monokine that acts systemically to alter body temperature, activate T cells, and induce hepatic protein synthesis of acute-phase reactants.

We and others have previously reported that human blood mononuclear cells release in culture certain substances that enhance the capacity of purified human blood eosinophils to kill the antibody-coated larvae of Schistosoma mansoni. The present study shows that this eosinophil cytotoxicity-enhancing activity (ECEA) is released by monocytes and T lymphocytes. Monocytes produce ECEA in resting and in LPS-stimulated cultures; T lymphocytes release such activity when stimulated by mitogens such as concanavalin A. Furthermore, the human monocytic line U-937 also releases ECEA-like activity when stimulated by LPS. The enhancing activity produced by monocytes has been partially characterized: it is sensitive to proteolysis by trypsin, relatively heat stable, and associated with molecules that have an apparent molecular weight of 14,000 to 65,000 daltons and isoelectric points of 3.8-3.9, 4.2, 4.5, 4.8-4.9. This shows that while ECEA produced by monocytes is heterogeneous in size and charge, it is probably different from interleukin 1.

Supernatants from the human continuous cell line, CM-S, have been tested for Interleukin-1 (IL-1) activity and found to constitutively elaborate this factor. The CM-S-produced IL-1 activity has been partially purified and shown to be similar to IL-1 produced by human and mouse peripheral blood macrophages. To the best of our knowledge this is the first continuous human cell line reported that produces this monokine.