Suppression of IL-12 production by phosphodiesterase inhibition in murine endotoxemia is IL-10 independent

Limbic system-associated membrane protein (LAMP), a 64-kDa membrane protein, is an axon guidance adhesion molecule expressed by neurons in limbic system-related areas of the CNS. During development, LAMP is expressed on growing axons, growth cones, and their target neurons, but in adults it is restricted to membranes of somata and dendrites. In the adult spinal cord, LAMP immunoreactivity is found only on neurons of lamina II, lamina X, and the intermediolateral cell column and its ultrastructural localization is entirely postsynaptic. We studied changes in the expression of LAMP in lamina II of adult rat spinal cord after L1-S2 dorsal rhizotomy, a procedure that partially deafferents lamina II neurons and induces axonal sprouting by spared systems in lamina II. At the light microscopic level, LAMP immunoreactivity in lamina II was decreased in density at 3, 10, and 60 days postoperatively. This decrease in immunoreactivity suggests that LAMP expression by lamina II neurons may normally be regulated by specific afferent activity. Ultrastructurally, in control lamina II and after deafferentation in both control and deafferented lamina II at 3 and 60 days postoperatively, LAMP expression was restricted to postsynaptic membranes. Ten days after deafferentation, however, when axons are actively sprouting, LAMP was expressed on both axonal and postsynaptic membranes. The reexpression of LAMP on axonal profiles after deafferentation may identify axons that undergo sprouting in response to deafferentation.     

Monocyte induction of IL-10 and down-regulation of IL-12 by iC3b deposited in ultraviolet-exposed human skin

CD11b+ monocytic/macrophagic cells (Mo/Mph), which infiltrate into skin after UV irradiation, play an important role in UV-induced immunosuppression. Because in mice, blockade of CD11b (iC3b receptor) on monocytes and depletion of its ligand, iC3b, reverses UV-induced immunosuppression, we asked whether iC3b is deposited in human skin after UV, and whether iC3b can modulate the cytokine profile of Mo/Mph. Immunofluorescence studies revealed that iC3b was newly deposited in UV-exposed skin and was localized in apposition to infiltrating CD11b+ Mo/Mph. In addition, in situ hybridization studies showed that TNF-alpha mRNA was also induced in a similar microanatomic localization. To model the effects of these complex signals on infiltrating Mo/Mph following UV exposure, we then tested the effects of immobilized iC3b and TNF-alpha on resting blood monocytes. Both IL-10 mRNA synthesis and protein secretion were significantly induced by binding of iC3b in vitro and were synergistically increased by the presence of TNF-alpha. The effect was abrogated by a blocking Ab to CD11b, indicating CD11b-iC3b interaction. In contrast, iC3b binding resulted in suppression of IL-12 p40 mRNA and significantly inhibited the production of IL-12 p70 protein. Our studies thus define a novel mechanism for induction of tissue Mo/Mph into an IL-10high/IL-12low state via iC3b in combination with TNF-alpha.     

Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro

Antigen-presenting cells are thought to modulate the development of Th1 and Th2 cells by the secretion of interleukin-10 (IL-10) and IL-12. Because glucocorticoids (GC) favor the development of Th2 responses, we determined whether dexamethasone (DEX) and hydrocortisone (HC) have differential effects on lipopolysaccharide-induced IL-10 and IL-12 production in whole-blood cultures. Significant inhibition of IL-12(p40) and IL-12(p70) was found with 10(-8) mol/L and 10(-9) mol/L DEX respectively, whereas IL-10 was relatively insensitive or even stimulated. Accordingly, the expression of IL-12(p40) and IL-12(p35) mRNA was more sensitive to DEX than IL-10 mRNA. The glucocorticoid receptor (GR) antagonist RU486 enhanced IL-12 production and largely abrogated the inhibition of IL-12 by GC, indicating that this suppression was mainly GR-mediated. High concentrations of RU486 were inhibitory for IL-10, suggesting that GC may exert a positive effect on IL-10. In the presence of neutralizing anti-IL-10 antibodies, DEX was still capable of IL-12 suppression whereas RU486 still enhanced IL-12 production, indicating that GC do not modulate IL-12 via IL-10 exclusively. Taken together these results indicate that GC may favor Th2 development by differential regulation of IL-10 and IL-12.     

Dose-dependent induction of IL-12 but not IL-10 from human keratinocytes after exposure to ultraviolet light A

OBJECTIVE: Our purpose was to assess the risk of ectopic pregnancy among women who smoke cigarettes. STUDY DESIGN: We used data from a case-control study of ectopic pregnancy conducted from October 1988 to August 1990 at an inner-city hospital in Georgia. Cases were 196 non-Hispanic black women with a surgically confirmed ectopic pregnancy. Controls were non-Hispanic black women who had delivered either a live or a stillborn infant weighing at least 500 gm (n = 882) or who were pregnant and seeking an induced abortion (n = 237). RESULTS: After we adjusted for parity, douching history, history of infertility, and age, the odds ratio for ectopic pregnancy was 1.9 (95% confidence interval 1.4 to 2.7) for women who smoked during the periconception period compared with women who did not smoke at that time. After stratification by the amount of daily smoking during the periconception period, the odds ratio rose from 1.6 (95% confidence interval 0.9 to 2.9) for women who smoked 1 to 5 cigarettes to 1.7 (95% confidence interval 1.1 to 2.8) for women who smoked 6 to 10 cigarettes to 2.3 (95% confidence interval 1.3 to 4.0) for women who smoked 11 to 20 cigarettes, and to 3.5 (95% confidence interval 1.4 to 8.6) for women who smoked >20 cigarettes per day. CONCLUSION: In this inner-city population, cigarette smoking was an independent, dose-related risk factor for ectopic pregnancy among black women. The public health and medical care communities should inform the public of this additional risk associated with cigarette smoking and intensify intervention strategies to reduce cigarette smoking among women of reproductive age.     

Molecular mechanisms involved in the inhibition of neutrophil locomotion by tumor cells

A chronic myelogenous leukemia cell line (K562) releases a factor of about 8 kD which we have named K562-inhibitory factor (K562-IF) because it inhibits neutrophil locomotion. This factor has potent anti-inflammatory activity in mice, associated with an inhibition of neutrophil function including not only random locomotion and fMetLeuPhe- or serum-induced locomotion but also adherence and zymosan-induced chemiluminescence and degranulation. In contrast, K562-IF does not affect the oxidative burst induced by soluble compounds such as fMetLeuPhe and phorbol esters. Analysis of the mechanism of action of K562-IF on neutrophils showed that it involves an adherence protein, mainly CR3 (the receptor of complement fraction iC3b). Neither, CR3 expression nor its up-regulation were altered, whereas the function of CR3 was depressed, i.e., it failed to cap upon neutrophil stimulation and did not bind iC3b. One unexplained finding is that K562-IF inhibits actin polymerization induced by fMetLeuPhe but not by activation of the Fc-gamma receptor III. Studies are underway to establish whether K562 cells are representative of other malignant cells with regard to the production of neutrophil inhibitors.     

Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12

We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-alpha and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-alpha and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naive macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-gamma dose dependently and differentially regulated subsequent LPS-induced production of TNF-alpha, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-alpha and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-alpha responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-alpha response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-alpha production. We also found that the initial LPS priming of naive macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-gamma in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-alpha and NO responses in LPS-primed macrophages.     

Impaired production of IL-12 in systemic lupus erythematosus. I. Excessive production of IL-10 suppresses production of IL-12 by monocytes

Currently, primary osteoporosis is the most frequent metabolic disease in women after menopause [1]. The resulting loss of bone mass is accompanied by an increased risk of skeletal fragility. One reason for the development of osteoporosis might be an impaired function of mature osteoblasts. To evaluate the involvement of specific growth factors in bone remodeling, cell cultures of osteoblastic cells derived from nonosteoporotic and osteoporotic postmenopausal women were established. The influences of TGF beta-1 and IGF-I on proliferation and mRNA expression of TGF beta-1 were investigated by [3H]-thymidine incorporation and competitive RT-PCR. We found IGF-I to have no significant effect on proliferation in cells of osteoporotic and nonosteoporotic patients. In contrast, differences were found in TGF beta-1 mRNA expression after application of IGF-I. Application of TGF beta-1 enhanced its own mRNA expression in both groups in a similar manner. Whereas the proliferation of cells of nonosteoporotic patients was inhibited by (10(-10) M) TGF beta-1, this treatment led to an increased proliferation of cells of osteoporotic patients.     

IL-12 prevents neonatal induction of transplantation tolerance in mice

We investigated the effect of IL-12 on the induction of transplantation tolerance by neonatal injection of allogenic cells. We first observed that injection of newborn BALB/c mice with IL-12 and (A/J x BALB/c)F1 spleen cells prevented the Th2 alloimmune response induced by neonatal inoculation of F1 cells alone and allowed the differentiation of T cells secreting high amounts of IL-2 and IFN-gamma in mixed lymphocyte cultures with donor-type stimulators. Furthermore, IL-12 administration resulted in the emergence of anti-donor cytotoxic T lymphocyte responses although at lower levels than in control uninjected mice. In parallel, we found that mice injected at birth with IL-12 and F1 cells did not develop chimerism and were able to reject a donor-type skin graft as efficiently as control mice. We conclude that IL-12 inhibits the Th2 polarization of the newborn response to alloantigens and prevents thereby the establishment of transplantation tolerance.     

Differential stimulation of interleukin-12 (IL-12) and IL-10 by live and killed Helicobacter pylori in vitro and association of IL-12 production with gamma interferon-producing T cells in the human gastric mucosa

The objective of these experiments was to examine the ability of Helicobacter pylori to stimulate interleukin-10 (IL-10) or IL-12 and select for either Th1 or Th2 cells. Gastric biopsy specimens were collected from patients who were categorized with respect to the presence of H. pylori and gastric disease as well as their age, gender, medications, and other factors. As Th1 and Th2 cells are selected by IL-12 and IL-10, respectively, biopsy specimens were screened for mRNA and protein for these cytokines. Although mRNA for IL-12 and IL-10 was detected in biopsy specimens obtained from both infected and uninfected patients, IL-12 protein predominated. Levels of IL-10 and IL-12 in gastric tissue did not change in response to infection. Moreover, gamma interferon (IFN-gamma)-producing T cells were found in both the infected and the uninfected gastric mucosa. Stimulation of peripheral blood leukocytes from either infected or uninfected donors with various concentrations of live or killed H. pylori induced immunoreactive IL-12 and IL-10. After stimulation with live H. pylori, IL-12 levels increased more than 30-fold, whereas IL-10 levels increased only 2- to 5-fold, compared to cells stimulated with medium alone. Interestingly, killed H. pylori induced significantly more IL-10 (P < 0.05) than live H. pylori, while recombinant urease only induced IL-10. These results demonstrate that live H. pylori selectively stimulates the induction of IL-12 and Th1 cells that produce IFN-gamma, whereas preparations used in oral vaccines induce more IL-10 and may favor Th2 cell responses.     

Stimulation of beta-adrenoceptors inhibits endotoxin-induced IL-12 production in normal and IL-10 deficient mice

The influence of water activity (a(w)) on the kinetics of aflatoxin and zearalenone production in amaranth grains at 25 degrees C was studied. Minimum a(w) for aflatoxin production in this substrate was 0.825. Accumulation of the four aflatoxins (B1, B2, G1 and G2) was similar at a(w) 0.825 (maximum 81.2 microg/kg after 42 days) and 0.868 (maximum 109.6 microg/kg after 49 days). Maximum accumulation of total aflatoxins at a(w) 0.902 (260.4 microg/kg) was detected after 21 days, with an appreciable increment in the concentration of aflatoxins B1 and G1. These quantities were lower than those reported for aflatoxin production on other cereals and legumes, indicating that amaranth is not a good substrate for aflatoxin production. Zearalenone was not detected at a(w) 0.902. Maximum accumulation of zearalenone was 1.5 microg/g after 35 days at a(w) 0.925 and 11.1 microg/g after 49 days at a(w) 0.950.     

IL-12 is an effective adjuvant for induction of mucosal immunity

OBJECTIVE: To define those physiologic and clinical variables that have a positive or negative predictive value in discriminating survivors from nonsurvivors with traumatic injuries and a Trauma Score of 5 or less. METHODS: A retrospective review of 2,622 trauma patients transported by an air medical service from the scene of injury to a Level I trauma center was performed. Demographic, physiologic, and clinical variables were evaluated. RESULTS: One hundred thirty-six patients were studied; 14 patients survived trauma resuscitation. Survivors had statistically significant improvement in the Glasgow Coma Scale from the field to arrival in the emergency room. Revised Trauma Score, probability of survival, pulse, respiratory rate, cardiac rhythm, central nervous system activity, and signs of life were statistically more favorable in survivors. CONCLUSION: In patients who survived to discharge, signs of central nervous system activity in the field was a positive predictor of survival, and severe head injury served as a negative predictor of survival.     

Regulation of the IL-12 receptor beta2 subunit by soluble antigen and IL-12 in vivo

Sympathetic innervation of lymphoid tissues is localized to specific tissue compartments, but little is known of the "factors" that are important in establishing this pattern during development. Numerous studies have shown interactions of nerve growth factor (NGF) with the immune system, which may include modulation of immune innervation. We previously have shown that NGF transgenic mice, which overexpress NGF in skin and not immune tissues, have a dramatic hyperinnervation of splenic marginal zone and peripheral lymph node medulla and capsule. The purpose of the current studies was to determine if the presence of elevated NGF would alter immune system development and the process of sympathetic ingrowth. The results show that the splenic innervation in NGF transgenics gradually diverged from controls during the first two postnatal weeks, with the greatest change occurring between postnatal days 13 and 16 when the splenic organization was reaching the adult pattern. In contrast, the peripheral lymph nodes were hyperinnervated at an earlier age. mesenteric lymph nodes never diverged from the normal pattern. NGF levels in transgenic spleen were much higher than controls at postnatal days 1 and 2, when little innervation was present, and declined as the tissue matured, possibly because of NGF uptake by the ingrowing sympathetic fibers. This suggests that immune tissues are capable of concentrating NGF, which in turn may modulate the level of innervation by the sympathetic nervous system.     

Histamine potently suppresses human IL-12 and stimulates IL-10 production via H2 receptors

IL-12 and IL-10, respectively, stimulate Th1 and Th2 immune responses. The development of some allergic reactions, infections, and tumors are associated with excessive histamine production and a shift toward Th2 responses. Here we address the possibility that this association is causally linked, at least in part, to modulation of IL-12 and IL-10 production by histamine. We report that histamine dose-dependently inhibited the secretion of human IL-12 (p70) and increased the production of IL-10 in LPS-stimulated whole blood cultures. These effects of histamine were antagonized by cimetidine, an H2 receptor antagonist, but not by selective H1 and H3 receptor blockers, and were mimicked by an H2 receptor agonist. The effects of histamine on IL-12 and IL-10 secretion were independent of endogenous secretion of IL-10 or exogenous addition of IL-12, while Ro 20-1724, a phosphodiesterase inhibitor, potentiated the effects of histamine on IL-12 and IL-10 production, implicating cAMP in its actions. Similar modulatory effects of histamine on IL-12 and IL-10 production, which were reversed by the H2 antagonist cimetidine, were observed in PBMC and isolated monocytes stimulated by Staphylococcus aureus Cowan strain 1 and LPS, respectively. Thus, histamine, via stimulation of H2 receptors on peripheral monocytes and subsequent elevation of cAMP, suppresses IL-12 and stimulates IL-10 secretion, changes that may result in a shift of Th1/Th2 balance toward Th2-dominance. This may represent a novel mechanism by which excessive secretion of histamine potentiates Th2-mediated allergic reactions and contributes to the development of certain infections and tumors normally eliminated by Th1-dependent immune mechanisms.     

Cross regulation by IL-10 and IL-2/IL-12 of the helper T cells and the cytolytic activity of lymphocytes from malignant effusions of lung cancer patients

STUDY OBJECTIVE: Our previous report demonstrated that there was impairment of local cellular immunity with elevated interleukin-10 (IL-10) and undetectable IL-12 in neoplastic pleural effusion. These findings suggest that the local immune reactions favor the T-helper type 2 (Th2) pathway instead of Th1 pathway. The present study was designed to examine whether local cellular immunity could be manipulated by IL-2 and/or IL-12 treatment, and to determine their effect on the helper T-cell pathways and the cytolytic activity of the effusion-associated lymphocytes (EALs). DESIGN: Using malignant pleural effusions obtained from four patients suffering from adenocarcinoma of lung, we separated the tumor cells from the EALs with Ficol-Hypaque centrifugation, followed by Percoll density centrifugation. To test whether the cytolytic function of lymphocytes could be enhanced by culturing with IL-2 and/or IL-12, lymphocytes were incubated with recombinant IL-2 with/without IL-12 for 6 days. Following this, the tumoricidal activity was assessed in an overnight 5'chromium-release assay. Autologous tumor cells for measuring specific antitumor activity, Daudi cells susceptible to lymphokine-activated killer cells, and NK-susceptible K562 cells were used as target cells. MEASUREMENTS AND RESULTS: After treatment in vitro with IL-2, IL-12, or IL-2 plus IL-12, the Th pathway shifted from Th2 to Th1 type (increased gamma-interferon production). To further study the effect of cytokine treatment on the cytolytic activity of EALs, it was found that after 6-day culturing, the EALs failed to kill any of the three tumor targets, whereas the 6-day cultured peripheral blood lymphocytes (PBLs) gave low level of cytotoxicity against all three tumor targets. Stimulation with IL-2 alone partially restored the immunocompetence of EALs to kill the tumor targets. Stimulation with IL-12 alone showed no significant effect on their cytolytic activity. However, IL-12 synergized with IL-2 to increase the cytolytic activity of EALs and PBLs against autologous tumor targets. This synergistic effect was not found for Daudi cells and K562 cells. CONCLUSIONS: These results suggest that EALs activated with IL-12 in the presence of a low concentration of IL-2, which converted the EALs from Th2 pathway to Th1 pathway, could be an alternative source of antitumor effectors for adoptive immunotherapy of cancer.     

Down-regulation of fibrinogen biosynthesis by IL-4, IL-10 and IL-13

High levels of fibrinogen are recognized as an important vascular risk factor; however, it is not known if the increase of plasma fibrinogen is directly responsible for this risk, or is only a marker of vascular inflammation. To support this second hypothesis, Oncostatin M (OSM) is a potent stimulator of fibrinogen biosynthesis and induces smooth muscle cell proliferation. In the same way, we analysed whether interleukin-4 (IL-4), interleukin-10 (IL-10) or interleukin-13 (IL-13), which protect vessel walls from monocytes injuries leading to atherosclerosis, could influence fibrinogen biosynthesis. The two levels of regulation of fibrinogen biosynthesis were tested: firstly, the direct effect of these cytokines on fibrinogen production by the hepatoma cell line Hep G2, and secondly their effect on the secretion of hepatocyte stimulating factor (HSF) activity in the supernatant of lipopolysaccharide (LPS)-activated monocytes. IL-4 and IL-13 added to Hep G2 cells down-regulated both the increase of fibrinogen secretion induced by IL-6 and fibrinogen mRNA levels, this effect being more pronounced when Hep G2 were preincubated with the two cytokines before IL-6 addition. The effect of IL-10 was evidenced only on mRNA expression. IL-10 and IL-13 dose-dependently decrease HSF activity secreted by LPS-activated monocytes, whereas IL-4 had no effect. However, the three cytokines decreased HSF activity when monocytes were incubated with the cytokines before LPS activation. The effects of these cytokines on HSF activity are related to variations of IL-6 and OSM secretion. Our data strengthen the hypothesis that the fibrinogen level is a marker of vascular disease, since cytokines which have a protective vascular effect down-regulate fibrinogen production.     

Requirement for in vivo production of IL-4, but not IL-10, in the induction of proliferative suppression by filarial parasites

Loss of T lymphocyte proliferation and the emergence of a host response that is dominated by a Th2-type profile are well-established features of human filariasis. We have previously reported that adherent peritoneal exudate cells (PEC) from mice transplanted with adult Brugia malayi parasites suppress the proliferation of lymphocytes without blocking Ag-cytokine production in vitro. We now show that infection of mice with the infective larval (L3) stage of B. malayi generates a similar population of PEC. Suppressive cells are generated within 7 days of infection and mediate their effects through a nitric oxide-independent pathway. Both L3 and adult infection elicit high levels of host IL-4 whereas the microfilarial stage of the parasite induces IFN-gamma production and does not generate a similar form of suppression. Production of host IL-4 was necessary to allow the generation of suppressive PEC, given that IL-4-deficient mice implanted with adult parasites failed to induce proliferative block. However, IL-10-deficient mice implanted with adult parasites resulted in T cell suppression, indicating that IL-10 is not essential for the induction of hyporesponsiveness. Neither IL-4 nor IL-10 were directly responsible for ablating cellular proliferation in vitro, as the addition of neutralizing Ab to either cytokine did not reverse the proliferative block. Thus, IL-4 produced in vivo in response to filarial L3 and adult parasites is essential for the induction of proliferative suppression but is not itself the suppressive factor.     

Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-gamma-producing cells

The role of IL-12 role in regulating Th1/Th2 balance is attributed in part to the ability of this cytokine to induce IFNgamma production by NK and Th1 cells, which in turn promotes Th1 and inhibits Th2 development. In the present study, the requirement for IL-12 in the development of alloantigen-reactive Th1 was assessed by adding neutralizing anti-IL-12 Abs or the IL-12 receptor antagonist p40 homodimer to primary MLC. The resulting cell populations were assessed for Th1 development by measuring IFN-gamma production upon restimulation with alloantigens. While the addition of anti-IL-12 Abs to primary MLC did not influence subsequent cytokine production, addition of p40 homodimer markedly enhanced, rather than decreased, Th1 development. To determine which T cell population produced enhanced levels of IFN-gamma in response to p40 homodimer, CD4+ or CD8+ T cells were depleted from the MLC. While p40 homodimer was inhibitory to selected CD4+ Th1 development, it enhanced IFN-gamma production by CD8+ T cells. To test the in vivo relevance of these findings, mouse heterotopic cardiac allograft recipients were treated with either p40 homodimer, anti-CD8 mAb, or with both p40 homodimer and anti-CD8 mAb. Treatment of allograft recipients with p40 homodimer had no effect on the in vivo sensitization of IFN-gamma-producing cells and resulted in accelerated allograft rejection relative to unmodified recipients. However, p40 homodimer markedly prolonged allograft survival in mice depleted of CD8+ T cells. Hence, p40 homodimer stimulates CD8+ Th1 development in vitro but inhibits CD4+ T cell function both in vitro and in vivo.