Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor

Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.     

Candida albicans double-stranded DNA can participate in the host defense against disseminated candidiasis

In the present work, we studied the in vitro immunomodulatory properties of double-stranded Candida albicans DNA and its protective effect in murine disseminated candidiasis. DNA induced the production of TNF-alpha by peritoneal macrophages and splenocytes in vitro through a chloroquine-dependent mechanism. Yeast DNA acted synergistically with IFN-gamma in triggering the secretion of nitric oxide by macrophages and enabled them to stimulate the proliferation of T cells in response to soluble anti-CD3. The effect of DNA on splenocytes is associated with an enhanced synthesis of IFN-gamma, IL-2 and IL-10. In vivo, DNA decreased the mortality and lowered the kidney contamination in mice intraperitoneally inoculated with C. albicans simultaneously with an increase in the specific proliferative response and cytokine production. The present results indicate that C. albicans DNA can provide protection against disseminated infection.     

Cytokine profile in mice with enhanced or depressed immune response to sheep erythrocytes induced by immunomodulator of bacterial origin--purified staphylococcal toxoid

Influence of immunomodulator of bacterial origin - purified staphylococcal toxoid (PST) - on the synthesisof proinlammatory (IL-1beta, IL-6, TNFalpha, IFN-gamma) and anti-inflammatory (IL- 10) cytokines, as well as cytokines directing the immune response to Th1 (IL-12) or Th2 (IL-4) type was studied in mice. Serum cytokines levels as well as levels of cytokines produced by splenocytes spontaneously or after stimulation by phytohemagglutinin were measured 4 and 24 hours after inoculation of PST. It was shown that PST in wide spectrum of doses (15; 1.5; 0.15 BU per mouse) was able to enhance or suppress synthesis of cytokines. Effect was nonlinear and its direction was depended from cytokine, time interval passed before obtaining the sample and dose of PST. For example, 15 BU of PST enhanced whereas 0.15 BU of PST suppressed the IL-6 production 4 hours after inoculation. Decrease of IL-6 level in serum 24 hours after inoculation of PST was detected. Synthesis of several serum interleukins (IL-2, IL-10) did not changed 4 and 24 hours after inoculation irrespective from dose of PST. It was demonstrated that modulation of humoral immune response in vivo induced by PST did not associated with modulation of cytokine profile. For example, increase of number of cells secreting antibodies to sheep erythrocytes was registered both during increased synthesis of cytokines (4 hours, IL-1beta, IL-6, IL-12) and during period of its depression (IL-6, TNF-alpha, IFN-gamma), as well as during stable production of cytokines (IL-1beta, IL-6, IFN-gamma).     

Cytokine production by different population of macrophages following radiation or combined radiation injury

Male mice (CBA x C57BL6)F1 were used for the experiments throughout this study. The levels of spontaneous and LPS-stimulated cytokines production (IL-1 beta, IL-6 and TNF-alpha) by peritoneal, splenic, and bone marrow macrophages were evaluated by means of enzyme-linked immunosorbent assay at 3, 6, 24, 48 and 72 hours after irradiation alone or combined injury (irradiation + thermal burn). The results suggest that macrophages, harvested from the main mice hematopoietic organs (bone marrow, spleen), did not increase cytokines production within the first three days following the 7 Gy gamma-irradiation or combined injury. Peritoneal macrophages revealed a capacity to enhance IL-6 and IL-1 production versus normal healthy mice. There were no significant differences of cytokine-producing activity if macrophages were harvested from irradiated or combined injured mice.     

Quantitative aspects of lipopolysaccharide and cytokine requirements to generate nitric oxide in macrophages from LPS-hyporesponsive (<Emphasis Type="Italic">Lps</Emphasis><Superscript>d</Superscript>) C3H/HeJ mice

Due to a gene defect (Lps(d)), C3H/HeJ mice are known to be hyporesponsive to the immunobiological potential of lipopolysaccharide (LPS). We studied dose requirements for LPS, IFN-gamma, and cytokines TNF-alpha and IL-10 to produce nitric oxide (NO) in peritoneal macrophages (Mphi) from these animals. In contrast to the Lps(n) C3H/HeN mice, high concentrations of LPS (up to 5 microg/mL) or IFN-gamma (up to 5 ng/mL) by themselves were unable to activate NO production in C3H/HeJ Mphi. The failure to produce NO could not be overcome by addition of L-arginine or tetrahydropterin. The high-output NO biosynthesis was dose-dependently stimulated by combined administration of varying concentrations of IFN-gamma (50-5000 pg/mL) and LPS (approximately 1 ng/mL) or to a lesser extent by IFN-gamma plus TNF-alpha or TNF-alpha/IL-10. Formation of NO in C3H/HeJ MCO triggered by high concentration of LPS (approximately 1 microg/mL) given together with IFN-gamma (0.2-5 ng/mL) reached the values typical for Lps(n) C3H/HeN mice. While Mphi from C3H/HeN mice secreted TNF-alpha, IL-10, and IL-10 upon contact with a low dose of LPS (1 ng/mL), C3H/HeJ Mphi required high concentration of LPS (5 microg/mL) to enhance the secretion of the cytokines. Yet, this dose remained ineffective to stimulate IFN-gamma in Mphi from C3H/HeJ mice. It can be presumed that one of the important factors influencing their deficient ability to form NO is a failure of Mphi to produce IFN-gamma upon LPS contact.     

Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production

The capacity of 12 cytokines to induce NO2- or H2O2 release from murine peritoneal macrophages was tested by using resident macrophages, or macrophages elicited with periodate, casein, or thioglycollate broth. Elevated H2O2 release in response to PMA was observed in resident macrophages after a 48-h incubation with IFN-gamma, TNF-alpha, TNF-beta, or CSF-GM. Of these, only IFN-gamma induced substantial NO2- secretion during the culture period. The cytokines inactive in both assays under the conditions tested were IL-1 beta, IL-2, IL-3, IL-4, IFN-alpha, IFN-beta, CSF-M, and transforming growth factor-beta 1. Incubation of macrophages with IFN-gamma for 48 h in the presence of LPS inhibited H2O2 production but augmented NO2- release, whereas incubation in the presence of the arginine analog NG-monomethylarginine inhibited NO2- release but not H2O2 production. Although neither TNF-alpha nor TNF-beta induced NO2- synthesis on its own, addition of either cytokine together with IFN-gamma increased macrophage NO2- production up to six-fold over that in macrophages treated with IFN-gamma alone. Moreover, IFN-alpha or IFN-beta in combination with LPS could also induce NO2- production in macrophages, as was previously reported for IFN-gamma plus LPS. These data suggest that: 1) tested as a sole agent, IFN-gamma was the only one of the 12 cytokines capable of inducing both NO2- and H2O2 release; 2) the pathways leading to secretion of H2O2 and NO2- are independent; 3) either IFN-gamma and TNF-alpha/beta or IFN-alpha/beta/gamma and LPS can interact synergistically to induce NO2- release.     

Dietary conjugated linoleic acid decreased cachexia,macrophage tumor necrosis factor-alpha production,and modifies splenocyte cytokines production

The effect of conjugated linoleic acid (CLA) on macrophage functions were studied in vitro, in vivo, and ex vivo. In RAW macrophage cell line, CLA (mixed isomers) was shown to inhibit lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) production. Two CLA isomers, c9,t11 and t10,c12, were tested on RAW cells and it was found that the c9,t11 was the isomer responsible for the inhibition of LPS-induced TNF-alpha production. BALB/c mice were used to determine the effect of dietary CLA on body weight wasting and feed intake after LPS injection. CLA was protective against LPS-induced body weight wasting and anorexia. Plasma TNF-alpha levels after LPS injection were lower in the CLA group compared with the corn oil-fed control group 2 hr post-LPS injection. In a separate experiment, 30 mice were fed a CLA-supplemented diet or a corn oil-supplemented diet for 6 weeks and peritoneal resident macrophages were obtained for measuring TNF-alpha and nitric oxide production after in vitro exposure to interferon-gamma (IFN-gamma) and/or LPS. TNF-alpha production was not found to be different in peritoneal macrophages from mice fed the dietary treatments, but less nitric oxide was produced in macrophages from CLA-fed mice upon stimulation when compared with macrophages from control-fed mice. Splenocytes were also collected from the mice fed the dietary treatments and stimulated to produce cytokines in culture. Supernatant was used to run cytokine enzyme-linked immunoabsorbant assays. Interleukin-4 (IL-4) was decreased in CLA-fed mice when splenocytes were stimulated with concanavalin A (Con A) for 44 hr; however, IL-2 and the IL-2-to-IL-4 ratio were elevated.     

Stimulation of cytokine and nitric oxide production by acyclic nucleoside phosphonates.

Several antiviral acyclic nucleotide analogues activate expression of genes for cytokines, such as TNF-alpha, IL-10 in macrophages and IFN-gamma in splenocytes. This is an underlying mechanism for substantially enhanced production of nitric oxide generated by IFN-gamma. More lipophilic prodrugs, bis-POM-PMEA and bis-POC-PMPA, are cytocidal for macrophages and thus inhibit nitric oxide formation.     

Activation state alters the effect of dietary fatty acids on pro-inflammatory mediator production by murine macrophages

Studies investigating the effect of dietary fats on pro-inflammatory cytokine production by macrophages (M phis) have yielded conflicting results. We hypothesised that this may be due to the different capacities of the M phis studied commonly (resident, thioglycollate-elicited) to produce prostaglandin E(2)(PGE(2)) and leukotriene B(4)(LTB(4)) which inhibit and stimulate, respectively, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) production. To investigate this, male C57Bl6 mice were fed for 6 weeks on a low fat (LF) diet or on high fat diets which contained coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO) as the main fat source. Production of TNF-alpha, IL-1 beta, PGE(2)and LTB(4)by lipopolysaccharide-stimulated resident and thioglycollate-elicited (i.e. inflammatory) peritoneal M phis was measured. PGE(2)production by both inflammatory and resident M phis was significantly decreased by FO feeding. FO also decreased LTB(4)production by resident M phis compared with LF feeding. Production of both cytokines by inflammatory M phis decreased with increasing unsaturation of the high fat diets, such that cells from FO-fed mice showed significantly decreased production of TNF-alpha and IL-1 beta compared to those from mice fed on each of the other diets. In contrast, resident M phis from mice fed FO showed increased TNF-alpha production compared to those from CO-fed mice. Thus, FO feeding decreases production of TNF-alpha and IL-1 beta by inflammatory M phis and increases production of TNF-alpha by resident M phis, at least in comparison to some other dietary fats. These results indicate the mechanisms by which dietary fats exert their effects upon pro-inflammatory cytokine production are most likely different for resident and inflammatory M phis.     

Modulation of antigen-presenting functions in peritoneal macrophages and changes in production of several cytokines in mice caused by purified staphylococcal toxoid

With adaptive transfer method it has been shown that immunomodulator purified staphylococcal toxoid (PST) changed (stimulate or suppress) antigen-presenting function (APF) of mice peritoneal macrophages (MF) in vivo. This phenomenon was registered during assessment of ability of peritoneal MF to present heterologous (with regard to PST) antigen--sheep erythrocytes (SE). Modulation vector depended from time interval between PST and SE inoculations. Inoculation of PST 1.5 h before SE resulted in stimulation of APF. When SE were inoculated to donor mice 24 h after PST, suppression of APF was developed. Suppression of APF was observed along with suppression of proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) as well as B-lymphocytes growth factor (IL-4). When cytokine profile was assessed 4 h after PST injection, the suppression of synthesis of these cytokines was not observed. Production of IL-12 increased in 9-12 times in 4 and 24 h after PST injection.     

Analysis of immunomodulating cytokine mRNAs in the mouse induced by mushroom polysaccharides

The immunomodulating action of two mushroom antitumor polysaccharides, polysaccharide-protein complex (PSPC) and lentinan, was elucidated through analysing the expression profile of cytokines during a time course (0 h to 48 h) after their administration. Among the 5 cytokine genes, the induction of a marked increase in the mRNA levels of IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma and M-CSF by PSPC and lentinan was observed in the peritoneal exudate cells and splenocytes. However, the time point of their increased production was different after PSPC and lentinan administration.     

Essential involvement of CX3CR1-mediated signals in the bactericidal host defense during septic peritonitis

Cecal ligation and puncture (CLP) caused septic peritonitis in wild-type (WT) mice, with approximately 33% mortality within 7 days after the procedure. Concomitantly, the protein level of intraperitoneal CX3CL1/fractalkine was increased, with infiltration by CX3CR1-expressing macrophages into the peritoneum. CLP induced 75% mortality in CX3CR1-deficient (CX3CR1(-/-)) mice, which, however, exhibited a similar degree of intraperitoneal leukocyte infiltration as WT mice. Despite this, CX3CR1(-/-) mice exhibited impairment in intraperitoneal bacterial clearance, together with a reduction in the expression of intraperitoneal inducible NO synthase (iNOS) and bactericidal proinflammatory cytokines, including IL-1beta, TNF-alpha, IFN-gamma, and IL-12, compared with WT mice. Bactericidal ability of peritoneal phagocytes such as neutrophils and macrophages was consistently attenuated in CX3CR1(-/-) mice compared with WT mice. Moreover, when WT macrophages were stimulated in vitro with CX3CL1, their bactericidal activity was augmented in a dose-dependent manner, with enhanced iNOS gene expression and subsequent NO generation. Furthermore, CX3CL1 enhanced the gene expression of IL-1beta, TNF-alpha, IFN-gamma, and IL-12 by WT macrophages with NF-kappaB activation. Thus, CX3CL1-CX3CR1 interaction is crucial for optimal host defense against bacterial infection by activating bacterial killing functions of phagocytes, and by augmenting iNOS-mediated NO generation and bactericidal proinflammatory cytokine production mainly through the NF-kappaB signal pathway, with few effects on macrophage infiltration.     

Identification of rat IL-1beta, IL-2, IFN-gamma and TNF-alpha in activated splenocytes by intracellular immunostaining.

We have developed a sensitive three-step indirect immunofluorescence method to identify individual rat cells that produce cytokines including IL-1beta, IL-2, IFN-gamma and TNF-alpha. Cultured rat splenocytes were polyclonally activated to cytokine synthesis by mitogens such as lipopolysaccharide or a combination of a protein kinase C activator (phorbol 12-myristate 13-acetate) and a calcium ionophore (ionomycin). Careful selection of either antigen affinity-purified polyclonal or monoclonal cytokine-detecting antibodies combined with gentle fixation and permeabilization of the cells enabled discrimination of cytokine-producing cells based on distinct morphological staining criteria. Cells making IL-2, IFN-gamma and TNF-alpha could be identified by a characteristic, intracellular, rounded, juxtanuclear immunofluorescence signal. This staining pattern reflected the accumulation of the intracellularly processed cytokines in the Golgi organelle of producer cells. The staining of cells that synthesized IL-1beta, which is not transported intracellularly via the endoplasmatic reticulum-Golgi pathway, generated a different, but distinct and reproducible staining pattern, IL-1beta producing macrophages expressed intense nuclear immunofluorescence with additional reticular, cytoplasmic signals. Furthermore, the use of biologically neutralizing detecting antibodies against the cytokines under study prevented recognition of surface-stained target cells that had bound secreted cytokines by cytokine-specific receptors. This modified staining technology enabled analysis of the kinetic pattern and the frequency of cytokine-producing cells in cultures of rat splenocytes after various modes of polyclonal activation.     

Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver

It has previously been determined that erythroid cells of mice are capable of expressing such cytokines as interleukin (IL) 1 alpha and beta, IL-4, IL-6, interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta). It has been shown that glycophorin A(+) (GlA(+)) and antigen erythroblasts (AG-EB(+)) (both human erythroid cells of embryonic origin) are also capable of producing a series of cytokines such as IL-1 beta, IL-2, IL-4 and IL-6. The aim of this work was to study the capacity of erythroid cells from human embryonic liver to produce such cytokines as IFN-gamma, TGF-beta1, tumour necrosis factor alpha (TNF-alpha) and IL-10. The erythroid cells were isolated by means of antibodies specific to erythroblasts (GlA and AG-EB), as well as those from single erythroid colonies. The production level of some cytokines varies insignificantly under the action of erythropoietin (Epo) and quantitatively differs in GlA(+) and AG-EB(+) erythroid cells. Hence, the erythroid cells express IFN-gamma, TGF-beta1, TNF-alpha and IL-10. The erythroid cells could be involved through the production of these cytokines in the regulation of such processes as self-renewal, proliferation and differentiation of cells of other blood-forming sites.     

Quantitative role of p42/44 and p38 in the production and regulation of cytokines TNF-alpha, IL-1beta and IL-12 by murine peritoneal macrophages in vitro by concanavalin A

In the present study the quantitative role of p42/44 and p38 in the production of TNF-alpha, IL-1beta and IL-12 by murine peritoneal macrophages, in vitro, on treatment with Concanavalin A (ConA) has been investigated. Maximum expression/production of cytokines TNF-alpha, IL-1beta and IL-12 was observed after 16 h by RT-PCR and 24 h by ELISA, on in vitro treatment with ConA. To investigate the role of MAP kinases in the production of cytokines, pharmacological inhibitors of MAP kinases--PD98059, SB202190 and SP600125, were used. The expression of TNF-alpha, IL-1beta and IL-12 was down regulated in the presence of PD98059 and SB202190 in a dose dependent manner, suggesting the involvement of p42/44 and p38 in ConA induced production of TNF-alpha, IL-1beta and IL-12 by macrophages. It was observed that SP600125 did not have any effect on the expression of TNF-alpha, IL-1beta and IL-12. Using different combinations of MAPK inhibitors, it was found that 45% signal is conveyed via p42/44 and 25% via p38 in the production of these cytokines, by ConA treated macrophages while 30% signal passes through unidentified pathways.     

Roquinimex-mediated protection effect on the development of chronic graft-versus-host disease in mice is associated with induction of Th1 cytokine production and inhibition of proinflammatory cytokine production

Roquinimex is an immunomodulator that can effectively inhibit the development of several autoimmune diseases in animal models, but the mechanism is still unknown. In this study, we investigated the effect of roquinimex on chronic graft-versus-host disease (GVHD) in mice, a well-established model for human systemic lupus erythematosus (SLE). Oral administration of roquinimex significantly suppressed the development of proteinuria and ameliorated nephritis symptoms in chronic GVHD mice. In addition, renal histopathology and immunohistochemistry studies revealed reduced glomerulonephritis and decreased IgG deposition in chronic GVHD mice treated with roquinimex. Chronic GVHD is characterized by a predominance of Th2 cytokines, and proinflammatory cytokines that also play an important role in the pathology of tissue damage. Therefore, we focused on the effect of roquinimex on cytokine production. Chronic GVHD mouse splenocytes exhibited severely reduced interferon (IFN)-gamma production in response to Concanavalin (Con A) stimulation and an overt Th2 skewness. Roquinimex treatment, however, induced IFN-gamma production and restored the Th1/Th2 cytokine balance, although only a minimal effect of roquinimex on interleukin (IL)-4 secretion was observed. The production of the proinflammatory cytokines TNF-alpha and IL-1 beta by peritoneal macrophages from lipopolysaccharide (LPS)-treated GVHD mice was significantly inhibited by roquinimex treatment. These data suggested that the beneficial effect of roquinimex on lupus might, at least in part, result from a restoration of Th1/Th2 cytokine balance and inhibition of inflammatory cytokine production.     

Preexposure of macrophages to low doses of lipopolysaccharide inhibits the expression of tumor necrosis factor-alpha mRNA but not of IL-1 beta mRNA

LPS is known to be a potent activator of macrophages and induces the production of TNF-alpha and IL-1. However, the signaling events and regulatory mechanisms required for the activation of macrophages by LPS have not been resolved precisely. We show that LPS modulates its own response in macrophages. Proteose peptone-induced murine peritoneal macrophages (P-PEM) produce significant amount of TNF-alpha and IL-1 after stimulation with LPS. However, preexposure of macrophages to low doses (less than 1 ng/ml) of LPS renders them refractory to stimulation by a second round of LPS, as evaluated by production of TNF-alpha. The loss of sensitivity to a second round of LPS was selective for TNF-alpha production as the LPS-primed macrophages retained the ability to produce IL-1. Northern blot analysis was performed with total RNA obtained from control and LPS- (1 ng/ml) primed P-PEM after 3-h stimulation with a second round of LPS. The expression of TNF-alpha mRNA was inhibited in LPS-primed P-PEM, whereas the expression of IL-1 beta mRNA was the same in control and LPS-primed P-PEM, consistent with the data of biologic activities of these two cytokines. Zymosan-induced TNF-alpha production was the same in control and LPS-primed macrophages, indicating that not all of the pathways required for TNF-alpha production were affected by LPS priming. Monokines such as human (h) rIL-1 alpha, hrTNF-alpha, hrIL-6, and murine rIFN-beta could not substitute for the action of low doses of LPS, and addition of indomethacin could not restore TNF-alpha production. These results suggest that exposure of macrophages to low doses of LPS suppresses the production of TNF-alpha, but not of IL-1, by inhibiting the expression of mRNA through a noncyclooxygenase-dependent mechanism. Thus, LPS-induced production of TNF-alpha and IL-1 in macrophages are differently regulated.