Marija Brcic

poly(I:C) and LPS induce distinct IRF3 and NF‐κB signaling during type‐I IFN and TNF responses in human macrophages

Macrophages play major roles in the onset of immune responses and inflammation by inducing a variety of cytokines such as TNF and IFN‐β. The pathogen‐associated molecular pattern, polyinosinic‐polycytidylic acid [poly(I:C)], and LPS were used to study type‐I IFN and TNF responses in human macrophages. Additionally, activation of the key signaling pathways, IFN‐regulatory factor 3 (IRF3) and NF‐κB, were studied. We found that TNF production occurred rapidly after LPS stimulation. LPS induced a strong IFN‐β mRNA response within a short time‐frame, which subsided at 8 h. The IFN‐stimulated genes (ISGs), ISG56 and IFN‐inducible protein 10, were strongly induced by LPS. These responses were associated with NF‐κB and IRF3 activation, as shown by IRF3 dimerization and by nuclear translocation assays. poly(I:C), on the other hand, induced a strong and long‐lasting (>12 h) IFN‐β mRNA and protein response, particularly when transfected, whereas only a protracted TNF response was observed when poly(I:C) was transfected. However, these responses were induced in the absence of detectable IRF3 and NF‐κB signaling. Thus, in human macrophages, poly(I:C) treatment induces a distinct cytokine response when compared with murine macrophages. Additionally, a robust IFN‐β response can be induced in the absence of detectable IRF3 activation.

Toll-like receptor-4 is involved in eliciting an LPS-induced oxidative burst in neutrophils.

The lipopolysaccharide (LPS) receptor complex of mononuclear phagocytes is composed of Toll-like receptor-4 (TLR4), MD-2 and CD14. Other phagocyte populations may express similar LPS receptors. The transmembrane glycoprotein TLR4 was shown to induce or upregulate a variety of gene products, which collectively are the mediators of an LPS effect. In this study, an involvement of TLR4 in mediation of an oxidative burst was determined using murine peritoneal exsudate neutrophils and lucigenin-enhanced chemiluminescence (CL). The CL response was dependent on the LPS dose and the presence of serum, putatively a source of lipopolysaccharide-binding protein (LBP). In the absence of serum, a CL signal was elicited by 4 microg/ml LPS in peritoneal exsudate cells (PEC) from TLR4-sufficient (C3H/HeN) but not TLR4 deficient (C3H/HeJ) mice. The signal obtained in PEC from TLR4-sufficient mice was completely abrogated by superoxide dismutase (SOD), which indicated that the response depended on the formation of superoxide anion, and was also seen in purified neutrophils but not purified macrophages (Mphi). In the presence of serum, lower LPS concentrations (e.g. 40 ng/ml) elicited a strong CL response in PEC from TLR4-sufficient, and a weak signal in cells from TLR-4-deficient mice. This suggests that TLR4 engagement is involved in promoting an oxidative burst in murine neutrophils.

Transforming growth factor-beta and interleukin-10, but not interleukin-4, down-regulate procoagulant activity and tissue factor expression in human monocyte-derived macrophages

The effect of IL-4, IL-10, and TGF-beta on expression of procoagulant activity (PCA) and of surface-associated tissue factor (TF) by human monocyte-derived macrophages was determined. Monocytes were allowed to mature to macrophages in teflon bags, and were primed either in suspension cultures, or after subculturing in microtiter plates. PCA was determined in PBS-stimulated cells (constitutive PCA) or after stimulation with LPS for 6 hr. TGF-beta significantly reduced constitutive and LPS-induced PCA. This effect was associated with a reduction in surface-expressed TF, but was not correlated with TNF-alpha production in LPS-stimulated cells. The TGF-beta effect was seen both in suspension cultures and in adherent cultures. IL-10 strongly down-regulated LPS-induced PCA, an effect closely correlated with TNF production. It had a weaker, albeit significant effect on constitutive PCA, when tested on suspended cells, and PCA down-regulation was associated with reduction in TF surface expression. IL-4 reduced neither constitutive nor induced PCA in macrophages, and had little effect on TF surface expression, although it strongly down-regulated CD14 expression. Also in monocytes, IL-4 influenced TF expression to a lesser degree than IL-10 and TGF-beta. In the monocytoid cell line, THP-1, PCA/TF was down-regulated preferentially by TGF-beta. Our findings point to a complex cytokine-mediated regulation of PCA at the level of TF expression and possibly at additional levels.

Induction of nitric oxide synthase in bovine mononuclear phagocytes is differentiation stage-dependent.

Bovine monocytes and monocyte-derived macrophages (MDM) activated by various means were assessed for induction of inducible nitric oxide synthase (iNOS), using the Griess assay, Northern blotting and reverse transcription/polymerase chain reaction (RT-PCR). Interferon-gamma (IFN-gamma) induced little, if any, iNOS expression and NO production in MDM, although these cells responded to IFN-gamma in other regards. In contrasts, MDM produced copious amounts of NO when stimulated with LPS or Salmonella dublin, and this was paralleled by high steady state levels of iNOS mRNA. Heat-killed Listeria monocytogenes induced more iNOS mRNA and nitrite than IFN-gamma, but much less than L. mono-cytogenes and IFN-gamma combined. Monocytes differed from M phi with respect to iNOS induction and nitrite production in several regards: (i) LPS and S. dublin induced only low levels of iNOS mRNA and nitrite in monocytes, although cells responded to these stimuli in various other ways: (ii) IFN-gamma alone induced in monocytes iNOS mRNA generation and NO formation, although to a low and variable degree; (iii) upon maximal stimulation (e.g. by L. monocytogenes and IFN-gamma combined), monocytes produced much less nitrite than MDM, and mRNA levels were lower. Regulation of macrophage iNOS varies considerably between species. We provide the first evidence in any species that the steady state levels of iNOS mRNA and NO generation in monocytes and macrophages activated by various means depend on the stage of mononuclear phagocyte differentiation.

Effect of IgG for intravenous use on Fc receptor-mediated phagocytosis by human monocytes

Polyspecific IgG given intravenously at high doses (IVIG) is used for immunomodulatory therapy in autoimmune diseases such as idiopathic thrombocytopenic purpura and myasthenia gravis. It is assumed that the clinical effect is brought about in part by a modulation of mononuclear phagocyte function, in particular by an inhibition of Fc receptor (FcR) mediated phagocytosis. In the present study, the effect of IVIG on FcR‐mediated phagocytosis by monocytes was analysed in vitro. Since monocytes exposed to minute amounts of surface‐bound IgG displayed impaired phagocytosis of IgG‐coated erythrocytes (EA), the effect of IVIG was studied with mononuclear cells suspended in teflon bags in medium containing 10% autologous serum and IVIG 2–10 mg/ml). Monocytes pre‐exposed to IVIG and then washed, displayed impaired ingestion of EA when compared with control cells cultured in 10% autologous serum only. The decrease in phagocytosis was observed with sheep erythrocytes treated with either rabbit IgG or bovine IgGl and with anti‐D‐treated human erythrocytes. This suggests that phagocytosis via both FcR type I (FcRI) and type II (FcRII) was decreased. The impairment of phagocytosis was dependent on the presence of intact IgG and was mediated by IVIG from nulliparous donors and from multigravidae to the same extent, suggesting that alloantibodies contained in IVIG have a minor role in modulating FcR‐mediated phagocytosis by monocytes. A flow cytometric analysis using anti‐FcRI, FcRII and FcRIII monoclonal antibodies showed that IVIG treatment upregulated FcRI expression but did not significantly alter the expression of FcRII and FcRIII.

The response of HEK293 cells transfected with bovine TLR2 to established pathogen-associated molecular patterns and to bacteria causing mastitis in cattle ☆

Toll-like receptors (TLRs) are key sensors of pathogen-associated molecular patterns (PAMPs). Their role in immunity is difficult to examine in species of veterinary interest, due to restricted access to the knockout technology and TLR-specific antibodies. An alternative approach is to generate cell lines transfected with various TLRs and to examine the recognition of PAMPs or relevant bacteria. In this report, we examined whether recognition of various PAMPs and mastitis-causing bacteria is achieved by transfection of recombinant bovine TLR2 (boTLR2). Therefore, human embryonic kidney (HEK) 293 cells were transfected by whole boTLR2. A clonal analysis of stably transfected cells disclosed variable recognition of several putative TLR2 agonists although expressing similar amounts of the transgene and endogenous TLR6. One clone (clone 25) reacted by copious interleukin-8 (IL-8) production to several stimulants of TLR2 such as di-palmitoylated cysteyl-seryl-lysyl-lysyl-lysyl-lysine (Pam2), a biochemical preparation of lipoteichoic acid from Staphylococcus aureus, a commercial preparation of peptidoglycan from S. aureus, and heat-killed Listeria monocytogenes (HKLM). TLR2-dependent induction of IL-8 release was stronger in medium containing human serum albumin than in medium containing fetal calf serum. Clone 25 cells responded to high concentrations of S. aureus and to Escherichia coli causing mastitis, but not to Streptococcus uberis and to Streptococcus agalactiae which also cause mastitis. Stimulation by S. aureus was relatively weak when compared (i) with stimulation of the same cells by HKLM and PAMPs derived from S. aureus, (ii) with a clone stably transfected with TLR4 and MD-2 and stimulated by E. coli causing mastitis, and (iii) with interferon-gamma-costimulated bovine macrophages stimulated by S. aureus and S. agalactiae. Thus, clone 25 is suitable for studying the interaction of putative TLR2 agonists with bovine TLR2-transfected cells, provides a cell to search for TLR2-specific antibodies, and is a tool for studying the interaction of TLR2 with bacteria causing disease, e.g. mastitis, in cattle.

Inducible Nitric Oxide Synthase and Nitrotyrosine in Listeric Encephalitis: A Cross-species Study in Ruminants

Listeria monocytogenes (LM) is a Gram-positive facultative intracellular bacterium that causes fatal meningoencephalitis in humans and ruminants. A current paradigm predicts that intracellular bacteria are controlled by nitric oxide (NO) whose synthesis is catalyzed by inducible nitric oxide synthase (iNOS). The ability of macrophages (MΦ) to express iNOS shows extreme interspecies variability. Here the expression of iNOS and synthesis of NO was studied in listeric encephalitis of cattle, sheep, and goats. iNOS was expressed by a subset of MΦ in cerebral microabscesses in all three species. The level of iNOS expression and the density of cells per lesion expressing iNOS was highest in cattle, intermediate in sheep, and lowest in goats. The accumulation of nitrotyrosine (NT), an indicator of local NO synthesis, was observed in lesions of cattle but not in those of small ruminants. The density of iNOS-expressing cells in lesions was inversely correlated with the number of bacteria. No species differences were observed in regard to reactive oxygen intermediate (ROI) production by stimulated granulocytes, using the flow cytometric dihydrorhodamine-123 (DHR) method indicating ROI generation. Thus, the marked species differences in iNOS expression, NT accumulation, and LM content in lesions of ruminants with listeric encephalitis are explained by different amounts of ROI produced. It suggests that variations in the ability of MΦ to synthesize NO are of pathophysiological significance in listeriosis.

Increased IL-4 and decreased regulatory cytokine production following relocation of Icelandic horses from a high to low endoparasite environment

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but <or=10% of their offspring born in Europe (2nd generation) do so. Recently, we showed that PBMC from 1st generation horses produce more IL-4 than 2nd generation horses. Since helminths and allergens induce Th2 responses, we investigated whether horses domiciled in Iceland are Th2-biased, and whether this is determined by helminth infection. We compared the parasite burden and T cell responses between Icelandic horses living in Iceland or Switzerland. Horses in Iceland have higher faecal egg counts, higher tapeworm-specific IgG(T) levels and higher total serum IgE levels than horses in Switzerland. Nevertheless, horses in Iceland displayed a low proportion of IL-4-producing cells in PBMC cultures after polyclonal or parasite extracts stimulation. No IL-4-producing cells were found in PBMC from horses after stimulation by Culicoides extract. Addition of anti-IL-10 and anti-TGF-beta1 to PBMC cultures of horses in Iceland increased the proportion of IL-4-producing cells after polyclonal or parasite antigens stimulation but not stimulation with Culicoides extract. This paralleled the high levels of IL-10 and TGF-beta1 found in supernatants from PBMC cultures of horses in Iceland. Collectively, horses living in Iceland have a high parasite burden but low IL-4 production. This supports the hypothesis that heavy helminth infections have a suppressive effect on IL-4 production mediated by IL-10 and TGF-beta1.

Evidence for involvement of peptidoglycan in the triggering of an oxidative burst by Listeria monocytogenes in phagocytes

We have shown previously that in listeric encephalitis of cattle and rats, nitrotyrosine was produced in microabscesses, implying that both superoxide anion (O2–) and nitric oxide (NO) are present and react with each other. Evidence of local synthesis of NO by macrophages was provided, but the source of O2– remained unknown. Here we have examined whether phagocytes exposed to viable and heat‐killed Listeria monocytogenes (LMΔ) produce O2– and, if so, whether this results from direct interaction of phagocytes with the bacterial surface of L. monocytogenes or whether prior opsonization is required. Using lucigenin‐enhanced chemiluminescence (LCL) for the measurement of O2–, we show that LMΔ induces an oxidative burst in human neutrophils, monocytes and monocyte‐derived macrophages (Mφ). Viability is not required, and opsonization by antibodies and/or complement does not enhance the LCL signal. As Toll‐like receptors (TLR) were shown recently to mediate an oxidative burst, TLR agonists representative for pathogen‐associated molecular patterns (PAMPs) were tested for their ability to elicit an oxidative burst. These included lipoteichoic acid (LTA), bacterial peptidoglycan (PGN), recombinant flagellin, CpG‐containing DNA and double‐stranded RNA. Only PGN and flagellin consistently elicited an LCL signal resembling that induced by LMΔ with regard to the kinetics and cell spectrum stimulated. However, flagellin was unlikely to be responsible for the LMΔ‐mediated burst, as a flagellin‐deficient mutant showed no decrease in LCL. We therefore assume that in LMΔ, core PGN acts as a PAMP and directly induces an oxidative burst in all phagocyte populations. We conclude that in cerebral lesions superoxide anion is generated locally by phagocytes recognizing bacterial PGN.

Human macrophages respond to LPS in a serum-independent, CD14-dependent manner

Two crucial mediators of monocyte activation by lipopolysaccharide (LPS) are the acute phase plasma factor, lipopolysaccharide binding protein (LBP) and cell-surface-expressed CD14. Whether macrophage (M phi) recognized and respond to LPS in a similar manner is unknown. Here we show that human monocyte-derived M phi respond to LPS by tumor necrosis factor-alpha release and procoagulant activity upregulation by a similar dose response curve in the presence or absence of serum, suggesting that humoral factors such as LBP are relatively unimportant in the activation of M phi. Both serum-dependent and serum-independent activation of M phi by LPS require cellular CD14, as evidence by blocking studies with CD14-specific antibodies. Clones from the monocytoid cell line Mono Mac-6 selected for high LPS sensitivity displayed similar properties. When washed free of serum and cultured in the presence of calcitriol, they responded to LPS in a similar manner, regardless of the presence or absence of serum, and this response was inhibited by anti-CD14. It is hypothesized that during their differentiation. M phi acquire a functional substitute for the serum factor LBP, thereby being able to recognize low LPS concentrations in a milieu low in LBP concentration. It will be of interest to determine whether this is a high-affinity LBP receptor, LBP itself, or another cell surface constituent.