Failure of hydrocortisone to suppress the interferon-gamma-induced augmentation of interleukin 1 secretion of aged human monocytes

The combined effect of hydrocortisone (HC) and interferon-gamma and -alpha (IFN-gamma and -alpha) on human blood monocytes (Mo) interleukin 1 (IL 1) secretion was investigated. IL 1 was generated by treating the fresh or aged Mo with lipopolysaccharide (LPS), and quantitated by radioimmunoassay (RIA). Hydrocortisone, at the pharmacological attainable concentration of 10(-5) molar (M), markedly suppressed fresh Mo IL 1 secretion but had no effect at lower tested doses. Addition of IFN-gamma enhanced the IL 1 secretion of fresh Mo; however, the simultaneous addition of 10(-5) M HC and IFN-gamma resulted in marked suppression of the monokine release. Monocytes, when cultured in vitro for three days, lost the capacity to secrete IL 1. The loss of IL 1 secretory potential of aged Mo was prevented by preincubating them with IFN-gamma prior to LPS stimulation. IFN-alpha was ineffective in this regard. Aged Mo, pretreated with the combination of IFN-gamma and HC were still able to secrete abundant quantities of IL 1, demonstrating the failure of HC to suppress the IFN-gamma-induced augmentation of IL 1 secretory potential. Even suprapharmacologic doses of HC (10(-4) M) did not inhibit this enhancement and actually further augmented it. Thus, therapeutic concentrations of HC suppress IL 1 secretion of fresh Mo even in the presence of IFN-gamma; however, therapeutic or suprapharmacologic concentrations of HC do not inhibit the IL 1 secretory capacity of IFN-gamma-treated aged Mo.     

The human intracellular Mx-homologous protein is specifically induced by type I interferons

The murine Mx-1 protein is one of the best biochemically and functionally characterized interferon (IFN)-induced proteins that is necessary, and sufficient, for providing resistance to murine cells against viral influenza infection. Recently an intracellular human protein homologous to the murine Mx-1 protein has been identified by means of a specific monoclonal antibody. The restricted induction of this intracellular protein in human mononuclear cells (MNC) by various cytokines was investigated. MNC from 26 of 28 healthy people and 35 of 36 cancer patients before IFN-alpha therapy had no detectable Mx-homologous protein. Incubation of human MNC with IFN-alpha and IFN-beta for 24 h at different concentrations led to a dose-dependent induction of the Mx-homologous protein. All IFN-alpha or IFN-beta preparations tested were equally effective in eliciting this intracellular protein. IFN-gamma induced only 1% of the Mx amount elicited by type-1 IFN compared on a weight basis. Neither interleukin (IL) 1 nor IL3, IL4, IL5, IL6, tumor necrosis factor-alpha/beta, granulocyte colony-stimulating factor (CSF) or granulocyte macrophage-CSF at any of the concentrations tested were capable of eliciting any detectable amount of the Mx homolog, while IL2 was a poor Mx-homologous protein inducer. In the presence of high-titered IFN-alpha antisera both IL2 and IFN-gamma were unable to stimulate this protein, proving that IFN-gamma and IL2 indirectly induce the Mx homolog via IFN-alpha. Therefore, the human Mx-homologous protein is a strictly by type I IFN-regulated protein in human peripheral blood lymphocytes.     

Kinetics and characterization of interferon production by murine spleen cells stimulated with Legionella pneumophila antigens.

Formalin-killed Legionella pneumophila bacterial cells, as well as a purified cell wall preparation (designated F-1 antigen) containing lipopolysaccharide (LPS), stimulated production of interferons (IFNs) in mouse spleen cell cultures. L. pneumophila whole-cell vaccine induced an IFN that was pH 2 labile and neutralized by anti-IFN-gamma indicating that IFN-gamma was the dominant form present. F-1 antigen induced a mixture of IFNs, depending upon the age of the culture and cell types present. In freshly prepared whole-spleen cultures and in 2-h adherent cultures, F-1 induced predominantly IFN-alpha/beta. In whole-spleen cultures that were allowed to age for 24 to 48 h before stimulation, F-1 was seen to induce mostly IFN-gamma, with low levels of IFN-alpha/beta present. Since only IFN-alpha/beta was produced in T-cell-depleted populations (at 2 h or at 48 h), it is suggested that T cells are responsible for IFN-gamma production in aged cultures. Additionally, heat-treated F-1, Escherichia coli LPS, and heat-treated E. coli LPS all induced similar levels of IFN-gamma in whole-splenocyte or nonadherent cell cultures which were incubated 48 h before stimulation. This suggests that LPS present in F-1 is responsible for IFN-gamma production and that an activated cell population is required. These results show that L. pneumophila antigens can induce the production of various types of IFN in mouse spleen cell cultures through several mechanisms.     

Activation of human monocyte-derived macrophages cultured on Teflon: response to interferon-gamma during terminal maturation in vitro

Macrophages (M phi) are potential antitumor effector cells derived from circulating blood monocytes (mo). Most studies on human mo/M phi biology and function have been performed using immature mo precursor cells. However, the conclusions drawn may be questionable, as mo have to undergo terminal differentiation before they reach relevant tissue sites of inflammation and immune reaction. We have analyzed the ability of mo-derived, teflon-cultured M phi to respond to activating stimuli with an increased tumor cytotoxic effector cell function using recombinant interferon-gamma (IFN-gamma), IFN-alpha 2, granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin(IL) 2, IL 1 alpha, and bacterial lipopolysaccharides (LPS) as mediator molecules. It could be shown that the response of M phi to the most potent activator molecule, IFN-gamma, depends on the terminal differentiation from the mo stage to the mature M phi. Whereas adherent mo could be activated only moderately, M phi increased their cytotoxicity by a factor of up to 400. IFN-gamma activation positively correlated with the effector cell number, the time of incubation and the dosage used. Activation did not depend on the presence of LPS, and was lost within 24 to 48 h. LPS itself activated cells only in the microgram range. IFN-alpha 2 activated M phi only at a two log higher concentration than IFN-gamma; GM-CSF was only slightly effective, whereas M phi incubation with IL 1 alpha or IL 2 did not result in M phi activation. Thus, the ability of human M phi to become activated appears to be a function of cellular maturation and is acquired during the terminal step of M phi differentiation. Teflon-cultured M phi could facilitate studies of the activation of human M phi and may be more suitable cells for adoptive immunotherapy in cancer patients than blood monocytes.     

Effects of gamma interferon on release of tumor necrosis factor alpha from lipopolysaccharide-tolerant human monocyte-derived macrophages.

After an initial stimulation of human monocyte-derived macrophages with bacterial lipopolysaccharide (LPS), which produces substantial release of tumor necrosis factor-alpha (TNF-alpha), a subsequent exposure to LPS results in about an order-of-magnitude reduction in the levels of TNF-alpha released. We have shown that macrophages which have been stimulated with LPS and then maintained in culture without LPS for as long as 2 weeks do not regain their original capacity to secrete TNF-alpha upon a second LPS challenge. After 2 to 4 days in adherent culture, monocyte-derived macrophages which were not pretreated with LPS also experience a measurable decline in their capacity to release TNF-alpha in response to an initial LPS stimulation. When compared with these previously nonstimulated cells, however, the levels of TNF-alpha released by LPS-pretreated cells in response to a second LPS challenge decline by over 90% after 8 to 9 days in culture. Unstimulated cells spontaneously release barely detectable levels of TNF-alpha. In contrast to the release of TNF-alpha, unstimulated cells release significant levels of prostaglandin E2 continuously over time, and these levels are variably increased by no more than a factor of two in response to a single LPS stimulation. Prostaglandin E2 levels released by LPS-pretreated cells in response to a second LPS stimulation are much closer to the levels released by unstimulated cells. We have also demonstrated that gamma interferon (IFN-gamma) enhances TNF-alpha release from LPS-stimulated macrophages but not from phorbol myristate acetate-stimulated cells. Addition of IFN-gamma to macrophages either during the initial stimulation or during a second stimulation with LPS enhances levels of TNF-alpha released after the second LPS challenge. The greatest enhancement is observed when IFN-gamma is added during both exposures to LPS, but addition of IFN-gamma during only the initial LPS stimulation still results in marked enhancement of TNF-alpha release in response to a second stimulation with LPS 24 h later. If an interval of 2 days of culture in medium alone separates the first and second 24-h LPS stimulations, IFN-gamma enhances TNF-alpha release only when it is included during the second LPS exposure, indicating that, unlike the persistence of endotoxin tolerance, enhancement of TNF-alpha release by IFN-gamma is transient.     

Isotype switching in anti-immunoglobulin-activated B lymphoblasts: differential requirements for interleukin 4 and other lymphokines to elicit membrane vs. secreted IgG1

Anti-immunoglobulin (Ig)-activated B lymphoblasts, prepared by culturing high-density B cells with anti-Ig-Sepharose for 48 h, can be induced to secrete IgM and IgG1 by a mixture of T cell-derived lymphokines containing interleukin (IL) 4. In this study we have examined the conditions required for lymphokine-mediated induction of IgG1 secretion and membrane (m)IgG1 expression in B lymphoblasts. Resting B cells exposed to IL 4 (10-100 U/ml) during anti-Ig-mediated blast transformation did not secrete IgG1 upon subsequent culture with lipopolysaccharide (LPS) regardless of whether IL 4 was present or absent during the secondary culture. In contrast, B lymphoblasts previously exposed to IL 4 did secrete IgG1 in response to T cell-derived lymphokines [EL 4 supernatant depleted of IL 4; (D)EL 4 SN]. However, optimal IgG1 secretion was obtained when B lymphoblasts were simultaneously exposed to IL 4 and other lymphokines. Pre-exposure to (D)EL 4 SN, which contains IL 5 and IL 2, failed to prepare anti-Ig blasts to secrete IgG1 in response to LPS and IL 4. Inhibition of IL 5 and IL 2 activity in EL 4 SN suppressed IL 4-mediated IgG1 secretion. Together, these data indicate that B lymphoblasts require IL 5 and IL 2 in addition to IL 4 to secrete IgG1, and that the IL 4 signal(s) must precede or accompany those provided by the other lymphokines. As a measure of the fraction of cells capable of switching to IgG1, we assessed expression of mIgG1 on B lymphoblasts by fluorescence flow cytometry. B lymphoblasts cultured for 3 days with (D)EL 4 SN and IL 4 (10-100 U/ml) were 8% to 20% mIgG1+; in the absence of IL 4 blasts did not express detectable mIgG1. Although anti-Ig blasts treated with LPS and IL 4 did not secrete appreciable IgG1, a substantial fraction of B lymphoblasts (4% - 19%) cultured with LPS and IL 4, but not LPS alone, expressed mIgG1. These results suggest that LPS and IL 4 are sufficient to commit B lymphoblasts to mIgG1 expression, but that additional signals provided by T cell-derived lymphokines are required to elicit IgG1 secretion.     

Monocyte-derived dendritic cells release neopterin

Increased neopterin concentrations in body fluids are found in diseases associated with activated, cell-mediated immunity including infections, autoimmune diseases, and certain malignancies. Monocytes/macrophages are known to secrete large amounts of neopterin upon stimulation with interferon-gamma (IFN-gamma). Ontogenetically, the major part of dendritic cells (DC) belongs to the myeloid lineage. Therefore, we investigated whether cultured monocyte-derived DC can elaborate neopterin. Cells were treated with cytokines in the presence or absence of monocyte-conditioned medium as a maturation stimulus. DC secreted an average 3.5 nmol/l neopterin. In response to IFN-gamma, cells significantly increased their output of neopterin. In distinction to monocytes/macrophages, neopterin production in DC was highly sensitive to IFN-alpha and IFN-beta. Further, lipopolysaccharides (LPS) enhanced neopterin synthesis, whereas tumor necrosis factor alpha, interleukin (IL)-1beta, IL-2, IL-10, and IL-18 were ineffective. Simultaneously, tryptophan degradation by induction of indoleamine (2,3)-dioxygenase (IDO) was tested in stimulated cells. Our results showed that IFN-gamma as well as LPS are inducers of IDO in DC.     

Role and regulation of IL-12 in the in vivo response to staphylococcal enterotoxin B.

Injection of a staphylococcal superantigen (SAg) such as staphylococcal enterotoxin B (SEB) in adult mice results in cytokine production and cell proliferation which can lead to septic shock. The aim of the present work was to identify the cytokines and co-stimulatory molecules regulating the in vivo systemic release of IFN-gamma, a cytokine known to play an important role in the pathophysiology associated with bacterial infections. We demonstrate in this study that (i) in contrast to lipopolysaccharide (LPS), SEB administration induces high levels of the p70, bioactive form, of IL-12; (ii) IL-12 production in response to SEB requires both CD40-dependent signals and IFN-gamma secretion; (iii) the early systemic release of IFN-gamma (3 h post-treatment) in response to SEB is IL-12 independent, while the sustained, late response (6-9 h post-treatment) requires endogenous IL-12 production; (iv) IL-12 produced during the primary SEB response (day 0) is responsible for priming cells in vivo to high IFN-gamma production upon secondary challenge (day 2); (v) the priming effect of IL-12 is TCR unrelated, as SEB-primed animals secrete high levels of IFN-gamma in response to both staphylococcal enterotoxin A and LPS administered 48 h later. The ability of bacterial SAg to induce septic shock and to modulate the immune response to unrelated antigens may therefore be related to their unique capacity to induce systemic IL-12 production in vivo. These observations also help to explain why SEB-primed animals, known to express an anergic phenotype 48 h post-treatment (as judged by defective IL-2 production and proliferation), nevertheless display an increased capacity to secrete the inflammatory cytokine IFN-gamma.     

The effect of gamma interferon on IL-1 secretion of in vitro differentiated human macrophages

After being cultured overnight, human monocytes lose their ability to secrete interleukin-1 (IL-1) when stimulated by lipopolysaccharide (LPS). However, when these monocytes were cultured for up to 9 days with various concentrations of interferon-gamma (IFN-gamma), these cells were found to retain their ability to secrete appreciable amounts of IL-1 on LPS stimulation. However, the effect was observed only if the monocytes were exposed to the IFN before LPS stimulation and simultaneous addition of IFN and LPS to macrophages was ineffective. This effect of IFN-gamma was related to the concentration of IFN added to the cultures and was completely neutralized by a monoclonal antibody to IFN-gamma. In addition to inducing IL-1 secretion, IFN-gamma also appeared to increase the overall production of IL-1, since reinduction of IL-1 secretion was not associated with a decrease in intracellular IL-1 content. When these macrophages were initially cultured with IFN-gamma, washed, and further cultured with IFN free medium, these macrophages were found to progressively lose their capacity to secrete IL-1 in response to LPS. Conversely, when monocytes were initially cultured in medium free of IFN, washed, and then further cultured in new medium, but now containing IFN-gamma, these macrophages were found to regain their capacity to secrete IL-1. However, the amount of reinduced IL-1 secretion decreased as the length of the initial culture period without IFN increased, with less than optimal IL-1 secretion occurring if monocytes were allowed to mature for 6 days before IFN-gamma pretreatment. In summary, these studies suggest that IFN-gamma may be important in enhancing IL-1 production and secretion by maturing macrophages and tissue macrophages and consequently may play a role in regulating the accessory cell activity of these cells for a variety of immune responses in vivo.     

Tissue selectivity of interferon-stimulated gene expression in mice infected with Dam(+) versus Dam(-) Salmonella enterica serovar Typhimurium strains

The host interferon (IFN) system plays an important role in protection against microbial infections. Salmonella enterica serovar Typhimurium is highly virulent in the mouse model, whereas mutants that lack DNA adenine methylase (Dam(-)) are highly attenuated and elicit fully protective immune responses against murine typhoid fever. We examined the expression of IFN-responsive genes in several mouse tissues following infection with Dam(+) or Dam(-) Salmonella. Infection of mice with Dam(+) Salmonella resulted in the induction of host genes known to be indicators of IFN bioactivity and regulated by either IFN-alpha/beta (Mx1) or IFN-gamma (class II transactivator protein [CIITA] and inducible nitric oxide synthase [iNOS]) or by both IFN-alpha/beta and IFN-gamma (RNA-specific adenosine deaminase [ADAR1] and RNA-dependent protein kinase [PKR]) in a tissue-specific manner compared to uninfected animals. Since the Mx1 promoter is IFN-alpha/beta specific and the Mx1 gene is not inducible directly by IFN-gamma, these data suggest a role of IFN-alpha/beta in the host response to Salmonella infection. Mice infected with Dam(-) Salmonella showed reduced expression of the same set of IFN-stimulated genes (ISGs) as that observed after infection with wild-type Salmonella. The reduced capacity to induce ISGs persisted in Dam(-)-vaccinated mice after challenge with the virulent (Dam(+)) strain. Finally, although no Dam(-) organisms were recovered from the liver or spleen after oral infection of mice, ADAR, PKR, Mx, and CIITA expression levels were elevated in these tissues relative to those in uninfected mice, suggestive of the distant action of a signaling molecule(s) in the activation of ISG expression.     

Antigen-specific and nonspecific interferon response of peripheral blood leukocytes from patients with chronic brucellosis

Thirty seven blood samples from 21 patients with chronic brucellosis were studied for interferon response to Brucella specific antigens and to the classical IFN inducers. Whole blood technique for IFN induction and bioassay with A549 cells challenged with EMC virus for IFN detection were used. Two different antigen preparations (BRU-1 and BRU-2) used for the serologic diagnosis of brucellosis, stimulated significantly (P less than 0.001) the synthesis of IFN-alpha and IFN-gamma in the whole blood cultures from the patients with chronic brucellosis but not from the control subjects. The detoxified antigen (brucellin) was inactive as the IFN inducer. BRU-1 and BRU-2 antigens induced also low levels of IFN-alpha + IFN-gamma in the short term cultures of the separated peripheral blood leukocytes (5-10 X 10(6) cells/ml) from healthy blood donors. This resembled stimulation of the leukocytes with LPS. Brucellin was inactive in the leukocyte culture. Despite the chronic infection lasting many years the brucellosis patients had apparently intact IFN system because the response of their leukocytes to NDV, PHA + PMA or LPS was not significantly different from that of the healthy blood donors. The importance of the relative balance of the IFN system for the pathogenesis of brucellosis is suggested and contrasted with IFN disfunction in the acquired immune deficiency syndrome.     

Interferon-alpha2a is sufficient for promoting dendritic cell immunogenicity

Type I interferons (IFNs) are widely used therapeutically. IFN-alpha2a in particular is used as an antiviral agent, but its immunomodulatory properties are poorly understood. Dendritic cells (DCs) are the only antigen-presenting cells able to prime naive T cells and therefore play a crucial role in initiating the adaptive phase of the immune response. We studied the effects of IFN-alpha2a on DC maturation and its role in determining Th1/Th2 equilibrium. We found that IFN-alpha2a induced phenotypic maturation of DCs and increased their allostimulatory capacity. When dendritic cells were stimulated simultaneously by CD40 ligation and IFN-alpha2a, the production of interleukin (IL)-10 and IL-12 was increased. In contrast, lipopolysaccharide (LPS) stimulation in the presence of IFN-alpha2a mainly induced IL-10 release. The production of IFN-gamma and IL-5 by the responder naive T cells was also amplified in response to IFN-alpha2a-treated DCs. Furthermore, IL-12 production by IFN-alpha2a-treated DCs was enhanced further in the presence of anti-IL-10 antibody. Different results were obtained when DCs were treated simultaneously with IFN-alpha2a and other maturation factors, in particular LPS, and then stimulated by CD40 ligation 36 h later. Under these circumstances, IFN-alpha2a did not modify the DC phenotype, and the production of IL-10/IL-12 and IFN-gamma/IL-5 by DCs and by DC-stimulated naive T cells, respectively, was inhibited compared to the effects on DCs treated with maturation factors alone. Altogether, this work suggests that IFN-alpha2a in isolation is sufficient to promote DC activation, however, other concomitant events, such as exposure to LPS during a bacterial infection, can inhibit its effects. These results clarify some of the in vivo findings obtained with IFN-alpha2a and have direct implications for the design of IFN-alpha-based vaccines for immunotherapy.     

Effect of macrophage activation on killing of Listeria monocytogenes. Roles of reactive oxygen or nitrogen intermediates, rate of phagocytosis, and retention of bacteria in endosomes.

The role of macrophage activation in the killing of L. monocytogenes is unclear. Some studies suggest that activation for enhanced production of reactive oxygen and nitrogen intermediates may not be of central importance. Recent data have indicated an important role for interferon-gamma (IFN-gamma) induced retention of L. monocytogenes in endosomes. Data from the present study indicate that proteose peptone-elicited macrophages from DBA2/J, CD-1, and C3H/HeN mice are listericidal. Activation of these cells in vitro for 20 h by IFN-gamma (20 or 500 U/ml) increased H2O2 or nitrite production, but did not increase the number of L. monocytogenes killed during a subsequent 6-h or 7-h culture. Incubation of macrophages with IFN-gamma plus lipopolysaccharide (LPS) caused greater activation and increased the number of Listeria killed during a 6-h or 7-h culture. However, this seems primarily attributable to enhanced phagocytosis. Proteose peptone-elicited macrophages were significantly more effective than resident macrophages in preventing the escape of L. monocytogenes from endosomes into the cytoplasm. This capability was not significantly enhanced by IFN-gamma in vitro, but was enhanced by IFN-gamma plus LPS. This correlates well with the effects of these activation stimuli on killing of L. monocytogenes by proteose peptone-elicited macrophages. These results indicate that enhanced retention of L. monocytogenes in endosomes is induced by proteose peptone elicitation and that further macrophage activation in vitro by IFN-gamma does not improve listericidal activity.     

IFN-alpha2b increases interleukin-10 expression in primary activated human CD8+ T cells.

Interleukin-10 (IL-10) is a multifunctional cytokine with diverse effects on most hematopoietic cell types. It appears the principal function of IL-10 is to limit and ultimately terminate inflammatory response. We demonstrate here that interferon-alpha2b (IFN-alpha) increases the expression of IL-10 in activated primary CD8(+) T cells. Optimal induction of mRNA expression and protein synthesis was observed when IFN-alpha was added to cells activated by the combination of anti-CD3 monoclonal antibody (mAb) and IL-2. Maximal stimulation of IL-10 protein production was observed after prolonged incubation periods (48-72 h). No effects were observed on the production of IL-4, whereas IFN-gamma was produced with a faster kinetics than an untreated control. Our data indicate that IFN-alpha promotes the development of a CD8(+) T cell population with enhanced anti-inflammatory activity, which may play a critical role in the regulation of a proper immune response.     

Transforming growth factor-beta mRNA and protein in hypertrophic scar tissues and fibroblasts: antagonism by IFN-alpha and IFN-gamma in vitro and in vivo.

Hypertrophic scarring (HSc) following burn injury is a common, disfiguring, and functionally limiting form of dermal fibrosis, compromising recovery. Previously, elevated levels of transforming growth factor-beta1 (TGF-beta1), a fibrogenic cytokine, were found in wounds and serum of severely injured patients, antagonized in part by treatment with systemic interferon-alpha2b (IFN-alpha2b) both in vitro and in vivo. It is hypothesized that in wound healing after injury, platelets are an initial source of TGF-beta, but wound fibroblasts may be capable, after activation, of autoamplification of the initial response to injury by increasing TGF-beta mRNA and protein that may subsequently be responsive to IFN therapy with IFN-alpha or IFN-gamma or both. Using three pairs of site-matched HSc and normal fibroblasts from the same individuals, nonconfluent and near confluent fibroblasts were treated with TGF-beta, and cell proliferation and collagen production were assayed using cell counting and 18O2 isotopic uptake into hydroxyproline before analysis by gas chromatography-mass spectrometry (GC-MS). HSc and normal fibroblasts were assayed for the production of TGF-beta protein secretion using ELISA for TGF-beta1, TGF-beta2, and TGF-beta3 after acidification of medium samples from 96-h cultures. HSc and normal fibroblasts were treated with IFN-alpha2b or IFN-gamma or both for 96 h. Quantitative RT-PCR and Northern analysis were performed using newly synthesized internal standards for human TGF-beta1. TGF-beta stimulates both HSc and normal fibroblast proliferation. Collagen synthesis is greater in HSc than in normal fibroblasts and is maximally stimulated at 75 pM TGF-beta. TGF-beta stimulated collagen metabolism is antagonized by IFN-alpha or IFN-gamma or both in an additive fashion. HSc and normal fibroblasts not only possess the mRNA for TGF-beta1 but also secrete mature TGF-beta protein. Treatment of HSc and normal fibroblasts with IFN-alpha2b or IFN-gamma antagonizes TGF-beta protein production, and additive effects occur. RT-PCR demonstrates that after IFN treatment, downregulation of TGF-beta1 mRNA accounts in part for the reduction in protein secretion in HSc fibroblasts. Elevations of systemic TGF-beta may be due to wound fibroblasts. TGF-beta synthesis and antagonism of fibroblast TGF-beta protein secretion occurs with either IFN-alpha or IFN-gamma, in part by downregulation of TGF-beta1 mRNA levels.     

IFN-alpha and IL-18 exert opposite regulatory effects on the IL-12 receptor expression and IL-12-induced IFN-gamma production in mouse macrophages: novel pathways in the regulation of the inflammatory response of macrophages

We characterized the IL-12 response of mouse macrophages in terms of modulation of IFN-gamma production by cytokines (IFN-alpha and IL-18) and regulation of IL-12 receptor expression. Beta1 and beta2 IL-12R chain mRNA expression increased with time in culture in the absence of exogenous stimulation, with concomitant acquisition of responsiveness to IL-12 for IFN-gamma production. Expression of the IL-12R beta1 chain mRNA was increased further following IL-12 treatment as a consequence of IFN-gamma expression. IL-12 response was regulated differentially by IFN-alpha and IL-18. Neutralization of endogenous type I IFN increased IFN-gamma secretion, whereas exogenous IFN-alpha reduced it. In contrast, IL-18 enhanced IFN-gamma mRNA accumulation and IFN-gamma secretion in IL-12-stimulated, but not -untreated, cultures. The opposite effects exerted by IFN-alpha and IL-18 mirror their mutual capacity of regulating-in a negative or positive manner, respectively-the expression of the IL-12R beta1 chain. We suggest that differential regulation of IL-12 response by IFN-alpha and IL-18 can represent previously unrecognized regulatory mechanisms for maintaining suitable levels of differentiation/activation in macrophages.     

Selective induction of mononuclear phagocytes to produce neopterin by interferons

Interferon-gamma (IFN-gamma) has been shown to be a potent inducer of neopterin secretion by human peripheral blood monocytes/macrophages (1). In this paper, it is shown that other known stimuli of monocytes (e.g., to secrete proteases or to migrate) such as zymosan-activated human serum, lipopolysaccharide, human C3/iC3 and zymosan coated with complement were unable to trigger monocytes/macrophages to release neopterin. Monocytes/macrophages could be stimulated solely by IFN-gamma (25 U/ml) and IFN-alpha at very high concentrations (10,000 U/ml). In the case of human peripheral blood mononuclear cells (PBMNC), basically the same pattern was observed. If however, in the buffer controls PBMNC showed some neopterin release, all stimuli triggered an increase of neopterin secretion: 10,000 U/ml IFN-alpha induced the same amount of secreted neopterin as did 25 U/ml of IFN-gamma. Both caused higher levels of neopterin secretion than ZAS, LPS and C3/iC3. Amongst the supernatants from PBMNC, only those which were obtained from cells activated with IFN-gamma or -alpha stimulated monocytes/macrophages to produce neopterin. Supernatants from lymphocytes activated with zymosan, lipopolysaccharide and interferon did not contain neopterin, nor did the latter induce monocytes/macrophages to generate and secrete neopterin. Antibodies against IFN-gamma inhibited the triggering effect of the supernatants except when generated by IFN-alpha at 10,000 U/ml. These results demonstrate that both interferons, IFN-gamma and IFN-alpha, the latter only at a 400-fold higher concentration, can trigger monocytes/macrophages directly to secrete neopterin. ZAS, LPS and C3/iC3 are weakly effective only on a mixture of lymphocytes and monocytes/macrophages, provided this cell mixture shows already a basic spontaneous neopterin release.(ABSTRACT TRUNCATED AT 250 WORDS)