Modulation of nitric oxide, hydrogen peroxide and cytokine production in a clonal macrophage model by the trichothecene vomitoxin (deoxynivalenol)

Characterization of how vomitoxin (VT) and other trichothecenes affect macrophage regulatory and effector function may contribute to improved understanding of mechanisms by which these mycotoxins impact the immune system. The RAW 264.7 murine cell line was used as a macrophage model to assess effects of the VT on proliferation and the production of nitric oxide (NO), hydrogen peroxide (H2O2) and cytokines. Using the MTT cleavage assay, VT at concentrations of 50 ng/ml or higher was found to significantly decrease proliferation and viability of RAW 264.7 cells without stimulation or with stimulation by lipopolysaccharide (LPS) or interferon (IFN)-gamma. In the absence of an activation agent, VT (25-250 ng/ml) had negligible effects on the production of NO, H2O2, and cytokines. Upon activation with LPS at concentrations of 10 to 100 ng/ml, VT at 25-100 ng/ml markedly enhanced production of H2O2 but was inhibitory at 250 ng/ml. VT enhancement of H2O2 production was observed as early as 12 h after LPS stimulation. When IFN-gamma was used as the stimulant, VT (25-250 ng/ml) delayed peak H2O2 production. VT (25-250 ng/ml) also markedly decreased NO production in cells activated with LPS or IFN-gamma. Interestingly, VT superinduced TNF-alpha and IL-6 production in LPS-stimulated cells and also elevated TNF-alpha in IFN-gamma stimulated cells. These results suggest that VT can selectively and concurrently upregulate or downregulate critical functions associated with activated macrophages.     

Bidirectional signaling between Yersinia and its target cell

Lipopolysaccharide (LPS) exhibits a wide variety of bioactivities. Although it was generally proposed that the lipid A component represented the active center responsible for most of the bioactivities of LPS, a variety of lipid A partial structures and analogues were reported to have different properties. Lipopolysaccharide of the Re595 mutant of Salmonella minnesota is lack of O and part of the core polysaccharide (2 keto-3-deoxyoctanate (KDO) left on lipid A). Re595 lipid A (LA) and Re595 monophosphoryl lipid A (MPLA) differ in structure from Re595 LPS by lacking KDO and KDO plus phosphoryl group respectively. Whether these lipid A-common Re595 LPS preparations differed in activities, we investigated their effects on nitric oxide (NO), TNF-alpha, IL-6, and IL-12 induction from murine macrophage cell line RAW 264.7. RAW 264.7 cells (2 x 10(5) cells ml(-1)) were stimulated with these LPS preparations at 1 microg ml(-1) for 48 h. Re595 LPS, Re595 LA and Re595 MPLA significantly induced NO, TNF-alpha and IL-6 production; NO, TNF-alpha and IL-6 inducing capacities were in the order of LPS = LA > MPLA, LPS = LA = MPLA, and LPS = LA > MPLA respectively. However, these preparations did not induce IL-12 production from RAW cells even when stimulated in combination with IFN-gamma (20 U ml(-1)). IFN-gamma itself also induced NO, TNF-alpha and IL-6 production from RAW 264.7 cells. When RAW 264.7 cells were stimulated with IFN-gamma plus any of these preparations, effects were additive and synergistic for NO and IL-6 responses respectively. But TNF-alpha responses of RAW cells against these preparations were almost equal when cultured alone or in combination with IFN-gamma. Pre-treatment of RAW cells either with LPS, LA or MPLA at low concentration (0.1 microg ml(-1)) for 60 min before pulsing with IFN-gamma (20 IU ml(-1)) plus LPS (1 microg ml(-1)) for an additional 48 h, significantly (P < 0.01) decreased NO response. Although to a lesser extent, TNF-alpha and IL-6 responses were also decreased. Complete inhibition of NO inducing effect of these LPS preparations was achieved with polymyxin B at 40 microg ml(-1). But the concentration of polymyxin B to get a significant (P < 0.05) inhibitory effect on LPS was four times higher than that for LA or MPLA. Unexpectedly, polymyxin B also inhibited INF-gamma-induced NO production from RAW cells in a dose-dependent fashion. These findings suggested that effect of LPS was dependent, at least in part, on both the LPS polysaccharide chain length and the hydrophilic portion of LPS. In addition, not only LPS but also LA and MPLA exert either enhancing or suppressive effects, depending on their concentrations and the timing of their addition with respect to co-stimulators.     

Interleukin-4 and -13 inhibit tumor necrosis factor-alpha mRNA translational activation in lipopolysaccharide-induced mouse macrophages

The production of tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide (LPS)-stimulated macrophages can be markedly inhibited by the two closely related cytokines, interleukin (IL)-4 and IL-13. To investigate the molecular mechanism of this inhibition, we analyzed the effect of the two cytokines on TNF-alpha production and TNF-alpha mRNA accumulation in the mouse macrophage cell lines RAW 264.7 and J774 stimulated by LPS. Whereas LPS-induced TNF-alpha production is strongly suppressed by both cytokines, TNF-alpha mRNA accumulation is not significantly affected, indicating that IL-4 and IL-13 induce a translational repression of TNF-alpha mRNA. Transfection of reporter gene constructs containing different regions of the TNF-alpha gene revealed that the inhibitory action of IL-4 and IL-13 is mediated by the UA-rich sequence present in the TNF-alpha mRNA 3'-untranslated region.     

Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin

OBJECTIVES: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion. DESIGN: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct. SETTING: Basic science research laboratory. MAIN OUTCOME MEASURES: Secreted TNF-alpha protein concentration. RESULTS: After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct. CONCLUSIONS: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.     

Structure-activity relationships of lipopolysaccharide (LPS) in tumor necrosis factor-alpha (TNF-alpha) production and induction of macrophage cell death in the presence of cycloheximide (CHX) in a murine macrophage-like cell line, J774.1

The structure-activity relationships of lipopolysaccharide (LPS) in tumor necrosis factor-alpha (TNF-alpha) production and induction of macrophage cell death in the presence of cycloheximide (CHX) were examined in a murine macrophage-like cell line, J774.1. TNF-alpha production is one of the characteristic phenotypes of LPS-activated macrophages, and is observed upon incubation with LPS. On the contrary, macrophage cell death, which had been found in our laboratory (Amano F., Karahashi H., J. Endotoxin Res., 3, 415423 (1996)) and was examined as the release of lactate dehydrogenase (LDH) from cells into the culture supernatant, was observed 2.5 h after the addition of LPS in the presence of CHX. However, both TNF-alpha production and macrophage cell death were similarly dependent on the structures of LPS and lipid A. At more than 10 ng/ml, wild-type LPS from E.coli and S. minnesota exhibited the highest activity, and LPS as well as diphosphoryl lipid A from S. minnesota rough mutants also exhibited activity, but E. coli LPS detoxified by alkaline treatment or monophosphoryl lipid A from S. minnesota exhibited no activity even at 100 ng/ml. These results suggest that LPS-induced macrophage cell death in the presence of CHX shows similar requirements for LPS and/or lipid A structures as for the macrophage activation at higher doses than 10 ng/ml, although the former was not observed at 1 ng/ml LPS, while the latter was. However, TNF-alpha does not seem to be involved in the induction of macrophage cell death, because a neutralizing anti-TNF-alpha antibody failed to block the macrophage cell death and because recombinant TNF-alpha had little effect on the extent of LDH release in the presence or absence of LPS and/or CHX, and also because TNF-alpha production by LPS was abolished in the presence of CHX.     

Modulation of lipopolysaccharide-induced macrophage gene expression by Rhodobacter sphaeroides lipid A and SDZ 880.431

Rhodobacter sphaeroides lipid A (RsDPLA) and SDZ 880.431 (3-aza-lipid X-4-phosphate) are prototypic lipopolysaccharide (LPS) antagonists. Herein, we examined the ability of these structures to regulate murine macrophage tumor necrosis factor (TNF) secretion and LPS-inducible gene expression (tumor necrosis factor alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], IP-10, type 2 TNF receptor [TNFR-2], D3, and D8 genes). We report that RsDPLA alone (> 1 microgram/ml) induced low levels of TNF-alpha secretion and a selective pattern of gene expression in peritoneal exudate macrophages; SDZ 880.431 alone was completely inactive. When LPS was present at a low concentration (1 ng/ml), RsDPLA and SDZ 880.431 blocked TNF secretion and gene induction in a concentration-dependent fashion. In general, gene induction was measurably reduced by 10 to 30 ng of RsDPLA per ml or 300 ng of SDZ 880.431 per ml, but inhibition could be uniformly overridden by increasing the concentration of LPS. Although induction of all six genes by LPS was suppressed by either inhibitor, effective inhibitor concentrations depended on the gene of interest. Induction of TNFR-2 by LPS was relatively resistant to inhibition by RsDPLA, and induction of TNFR-2 and D3 was relatively resistant to inhibition by SDZ 880.431. When LPS was present at > or = 100 ng/ml, correspondingly high concentrations (> or = 20 micrograms/ml) of either inhibitor influenced gene expression in a bidirectional manner. Under these conditions, LPS-induced expression of IP-10, D3, and D8 was suppressed regardless of the LPS concentration used (concentrations tested up to 50 micrograms/ml), while expression of TNF-alpha mRNA was enhanced about fourfold. In toto, RsDPLA and SDZ 880.431, when present at low concentrations, act in a manner consistent with competitive inhibition of LPS, while at higher concentrations, these structures inhibit certain LPS responses noncompetitively and synergize with LPS for other responses.     

Suppressive effect of hepatitis B virus on the induction of interleukin-1 beta and interleukin-6 gene expression in the THP-1 human monocytic cell line

The major target organ for Hepatitis B Virus (HBV) is the liver, but extrahepatic sites including monocytes express receptors for HBV and may become infected. Therefore, we investigated the effect of HBV on the in vitro expression of interleukin-beta (IL-1 beta) and interleukin-6 (IL-6) by the monocytoid cell line THP-1, exposed to various stimuli (LPS, PMA or both). Nonstimulated THP-1 cells did not synthesize IL-1 beta and IL-6, even after in vitro exposure to HBV. LPS stimulation alone induced moderate secretion of both IL-1 beta and IL-6 (300 pg/ml). After induction of macrophage differentiation by PMA, THP-1 cells acquired adherence and expressed a higher level of IL-1 beta (up to 2 ng/ml) but did not synthesize IL-6. Treatment of THP-1 cells with PMA and LPS caused the highest production of both IL-1 beta and IL-6 (> 5ng/ml). In vitro exposure of PMA + LPS-stimulated THP-1 cells to HBV resulted in secretion of both HBsAg and preS2Ag which was maintained over 10 days of culture. Southern blot technique was used to study the state of HBV DNA in the cells. Hybridization of non-digested cellular DNA showed only high molecular weight HBV DNA forms. The HindIII restriction pattern revealed bands corresponding to large DNA fragments and the presence of bands at the 3.2 kb position. Under these conditions (PMA + LPS), HBV inhibited the production of IL-1 beta and IL-6 proteins and completely suppressed the IL-1 beta and IL-6 mRNA. Thus, our findings (i) strongly support a relationship between the state of cell differentiation and susceptibility of cells to HBV infection, and (ii) demonstrate that HBV exerts an inhibitory effect on the induction of IL-1 beta and IL-6 genes expression in monocytic THP-1 cells. These results suggest that HBV leads to a fall of pro-inflammatory cytokine production by monocytes/macrophages, which may contribute to impaired host immune response during infection.     

Expression of interleukin-3 and tumor necrosis factor-beta mRNAs in cultured microglia

The function of interleukin-3 (or multi-CSF) in the hemopoietic system has been studied in great detail. Although its growth promoting activity on brain microglial cells has been confirmed both in vitro and in vivo, its presence in the brain and even in cultured brain cells has repeatedly been questioned. We have shown recently that isolated rat microglia express mRNA(IL-3) and synthesize IL-3 polypeptide. It is shown here by use of the PCR method, that mRNA(IL-3) is found also in C6 glioblastoma, in rat aggregate cultures, and in newborn and adult rat brain. Quantitation of amplified cDNA(IL-3) was achieved by non-competitive RT-PCR using an elongated internal standard. IL-3 messenger RNA was almost undetectable in vivo and low in (serum-free) aggregate cultures. In isolated microglia, mRNA(IL-3) was increased upon treatment with LPS, PHA, with the cytokines IL-1 or TNF-alpha, with retinoic acid, dbcAMP or the phorbol ester TPA. Effects of LPS were inhibited by dexamethasone, while the glucocorticoid by itself had no effect on basal IL-3 expression. LPS increased mRNA(IL-3) in a concentration-dependent manner beginning with 10 pg/ml and reaching plateau levels at 10 ng/ml. LPS also increased mRNAs of TNF-alpha and TNF-beta. TNF-alpha mRNA was already detectable in untreated microglia and LPS-increased levels were sustained for a few days. In contrast, TNF-beta mRNA was observed only between 4 and 16 h of LPS incubation. It was absent in LPS-free microglia, and after 24 h of LPS-treatment or later.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcitriol modulates in vivo and in vitro cytokine production: a role for intracellular calcium

Calcitriol modulates in vivoand in vitro cytokine production: A role for intracellular calcium. Background. Several immunomodulatory properties of calcitriol are currently known, however, only little information is available regarding the in vivo and in vitro effects of calcitriol on cytokine production in chronic renal failure. Methods. To study the in vitro effect of calcitriol on lipopolysaccharide (LPS)-induced cytokine production, peripheral blood mononuclear cells (PBMC, 2.5 ml/ml) from 12 chronic dialytic (HD), 15 undialyzed chronic renal failure (CRF) patients and 10 normal subjects (N) were incubated at 37 degrees for 12 hours with 100 ng of LPS (E. coli and P. maltofilia). Increasing doses of calcitriol from 10-10 to 10-9 M were added and cell associated TNF-alpha and IL-1beta were determined by immunoreactive tests after three freeze-thaw cycles. The intradialytic TNF-alpha and IL-1beta production were evaluated in vivo in 12 HD patients before and after three months of intravenous calcitriol treatment (6 microgram/week). Intracellular calcium [Ca++]i was determined on PBMC with a cytofluorimetric assay using FLUO-3 AM as the indicator. Results. In vitro, TNF-alpha increased from 3.6 +/- 1.9 pg/cell to 1797 +/- 337 in N, from 4.5 +/- 1.7 to 1724 +/- 232 in CRF and from 3.4 +/- 2.3 to 1244 +/- 553 in HD after the LPS stimulus. The production of TNF-alpha was inhibited by calcitriol in a dose-dependent manner [LPS + Vit.D3 100 ng, 2.9 +/- 2.1 in N, 3.7 +/- 1.9 in CRF and 3.4 +/- 1.7 in HD; LPS + Vit.D3 50 ng, 263 +/- 296 (N), 6.73 +/- 11 (CRF), 38 +/- 28 (HD); LPS + Vit.D3 25 ng = 873 +/- 583 (N), 325 +/- 483 (CRF), 588 +/- 507 (HD); LPS + Vit.D3 12.5 ng, 954 +/- 483 (N), 912 +/- 510 (CRF), 875 +/- 527 (HD)]. Comparable data were observed on IL-1beta production. In vivo, the intradialytic TNF-alpha increase (from 8.5 +/- 2.3 to 19 +/- 5.6 pg/2.5 x 106 cell) during hemodialysis was markedly reduced after calcitriol therapy (from 6.6 +/- 3.1 to 11 +/- 4.7). [Ca++]i decreased from 105 +/- 25 to 72 +/- 18 nM (P < 0.05) and a positive correlation between cytokine levels and [Ca++]i was found (r = 0.79; P < 0.001). Conclusions. The in vitro increase of cell-associated cytokine after LPS challenge was inhibited by calcitriol in a dose-dependent manner. These data suggest a possible in vivo modulatory effect of calcitriol therapy on cytokine production in hemodialysis.     

Mg2+ coordination in catalytic sites of F1-ATPase

Cytokines are hormone-like proteins which mediate and regulate inflammatory and immune responses. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) and inflammatory cytokines on regulation of interleukin-6 (IL-6) production by human gingival fibroblasts (HGF). The HGF cell lines used in this study, H-CL and F-CL, were established by the explant technique from healthy gingival tissue. Cultured cells were grown to confluency and incubated with various concentrations of LPS from Escherichia coli or Porphyromonas gingivalis or with the recombinant human cytokine tumor necrosis factor alpha (TNF-alpha), IL-1alpha, or IL-1beta. Culture supernatants were collected at various times and assessed for IL-6 production by enzyme-linked immunosorbent assay. Total RNA was isolated from the harvested cells and used to assess levels of IL-6 mRNA by the RNase protection assay. Both LPS preparations induced IL-6 production (1 to 4 ng of IL-6 per ml) by both HGF cell lines. Although TNF-alpha stimulated IL-6 production by HGF, > 10-fold-larger amounts were induced with IL-1alpha and IL-1beta. Furthermore, the addition of both IL-1alpha and TNF-alpha to cultured cells resulted in approximately 600- to 800-fold-higher levels of IL-6 than seen in control cultures, suggesting that these cytokines synergistically induced IL-6 production by HGF. IL-6 message in cultured cells was upregulated 20-fold by TNF-alpha, 1,000-fold by IL-1alpha and IL-1beta, and 1,400-fold by IL-1alpha plus TNF-alpha. IL-1alpha and TNF-alpha alone upregulate IL-6 production in a dose- and time-dependent fashion. The addition of IL-1alpha and TNF-alpha to cultured HGF cells resulted in a synergistic induction of IL-6 after 8 h of incubation and when greater than 10 pg of this combination per ml was used. Our studies show that inflammatory cytokines are hundreds of times more potent than LPS in stimulating IL-6 production by HGF.     

CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol

The antitumor agent, Taxol, shares with bacterial LPS the ability to activate murine macrophages, and its LPS-mimetic effects are blocked by LPS analogue antagonists. Since CD14 is central to the recognition of LPS by macrophages, we sought to examine a role for CD14 in the response to Taxol vs LPS. A comparison of responses of macrophages from wild-type mice with those from mice lacking CD14 due to a targeted disruption of the CD14 gene (CD14-deficient knockout (CD14KO)) revealed that like LPS, Taxol induces both CD14-dependent and -independent pathways of gene activation, although the CD14 dependency of Taxol stimulation is much less striking than that observed with LPS. The macrophage interaction with low concentrations of LPS (< or = 10 ng/ml) is largely CD14 dependent, as evidenced by the lack of induction of TNF-alpha, IL-1beta, and interferon-inducible protein-10 (IP-10) genes by CD14KO macrophages cultured in the absence of soluble CD14 (i.e., in autologous CD14KO -/- mouse serum). However, at high concentrations of LPS or Taxol, a CD14-independent pathway of activation is observed: this pathway leads to minimal IP-10 gene induction, even though induction of TNF-alpha and IL-1beta occurs. Measurements of TNF secretion followed a similar pattern to that observed at the level of steady state mRNA. These data suggest the existence of two pathways of activation by both LPS and Taxol: one that is CD14 dependent and leads to induction of TNF-alpha, IL-1beta, and IP-10 gene induction, and a CD14-independent pathway that results in the induction of TNF-alpha and IL-1beta, with minimal induction of IP-10.     

Mimosine blocks cell cycle progression by chelating iron in asynchronous human breast cancer cells

We have used severe-combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)-10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)-gamma from natural killer cells in vitro. This response was enhanced up to 15-fold in the presence of exogenous IL-2, but was completely ablated by addition of IL-10 with IC50 of less than 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12-14 days, whereas daily administration of IL-10 increased bacterial replication in the liver and spleen by up to 1000-fold resulting in death by day 4 post-infection. The immunosuppressive actions of IL-10 in vivo were also observed in immunocompetent BALB/c mice, whereas doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL-10, peritoneal macrophage monolayers were challenged with Listeria after preincubation with a panel of recombinant cytokines. IFN-gamma primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL-10 production, whereas granulocyte/macrophage colony-stimulating factor (CSF), macrophage CSF and also IL-4 enhanced macrophage IL-10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN-gamma during infection of SCID mice with Listeria inhibited TNF-alpha mRNA, but augmented expression of IL-10 mRNA in infected tissues. These results demonstrate that exogenous Il-10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL-10 versus TNF is differentially regulated by the cytokine environment of the macrophage.     

Effects of interferons on tumour necrosis factor alpha production from human keratinocytes

Interferons (IFNs) have been reported to have pleiotrophic effects including the ability to induce the production of other cytokines in several cell types. Tumour necrosis factor alpha (TNF-alpha) is pro-inflammatory cytokine a known to be produced by a variety of cells including human keratinocytes. In the present study, we sought to determine the effects of IFNs on TNF-alpha production from human keratinocytes. IFN-gamma (50-100 ng/ml) induced TNF-alpha production dose dependently, but no induction of TNF-alpha was observed with IFN-alpha or IFN-beta. Since in the epidermis cytokines often work with in a cascade fashion and keratinocytes are a source of primary cytokine, IL-1 alpha, whether combined treatment with IFN-gamma and IL-1 alpha had a synergistic effect on TNF-alpha production was examined. Combined treatment with IFN-gamma (100 ng/ml) and IL-1 alpha (10 ng/ml) induced 2-3-fold higher level of TNF-alpha than IL-1 alpha alone. These results suggest that IFN-gamma is a positive regulator for the production of TNF-alpha from human keratinocytes and likely to increase skin inflammation.     

Induction of cytokines by a phenylpropanoid glycoside acteoside

A phenylpropanoid glycoside acteoside was found to induce interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in macrophage-like cell line J774.A1 at 1-100 ng/ml. In addition, when the stimulatory action of acteoside was studied using the bovine glomerular endothelial cell line GEN-T, it stimulated IL-6 production. These stimulatory activities were not abolished by treatment with polymixin B, which inactivates lipopolysaccharide (LPS), indicating that the action was not a contamination of LPS.     

Selective suppression of interleukin-12 induction after macrophage receptor ligation

Interleukin (IL)-12 is a monocyte- and macrophage-derived cytokine that plays a crucial role in both the innate and the acquired immune response. In this study, we examined the effects that ligating specific macrophage receptors had on the induction of IL-12 by lipopolysaccharide (LPS). We report that ligation of the macrophage Fcgamma, complement, or scavenger receptors inhibited the induction of IL-12 by LPS. Both mRNA synthesis and protein secretion were diminished to near-undetectable levels following receptor ligation. Suppression was specific to IL-12 since IL-10 and tumor necrosis factor-alpha (TNF-alpha) production were not inhibited by ligating macrophage receptors. The results of several different experimental approaches suggest that IL-12 downregulation was due to extracellular calcium influxes that resulted from receptor ligation. First, preventing extracellular calcium influxes, by performing the assays in EGTA, abrogated FcgammaR-mediated IL-12(p40) mRNA suppression. Second, exposure of macrophages to the calcium ionophores, ionomycin or A23187, mimicked receptor ligation and inhibited IL-12(p40) mRNA induction by LPS. Finally, bone marrow-derived macrophages from FcR gamma chain-deficient mice, which fail to flux calcium after receptor ligation, failed to inhibit IL-12(p40) mRNA induction. These results indicate that the calcium influxes that occur as a result of receptor ligation are responsible for inhibiting the induction of IL-12 by LPS. Hence, the ligation of phagocytic receptors on macrophages can lead to a dramatic decrease in IL-12 induction. This downregulation may be a way of limiting proinflammatory responses of macrophages to extracellular pathogens, or suppressing the development of cell-mediated immunity to intracellular pathogens.     

The biphasic mRNA expression pattern of bovine interleukin-8 in Pasteurella haemolytica lipopolysaccharide-stimulated alveolar macrophages is primarily due to tumor necrosis factor alpha

Pasteurella haemolytica serotype 1 is the bacterial agent responsible for the pathophysiological events associated with bovine pneumonic pasteurellosis. Our previous studies support a role for the lipopolysaccharide (LPS) from P. haemolytica in the induction of proinflammatory cytokines. One of the pathological hallmarks of bovine pneumonic pasteurellosis is an influx of neutrophils into the alveolar spaces. This pronounced influx suggests the local production of a chemotactic factor(s) such as interleukin-8 (IL-8). In the context of the lung, the alveolar macrophage appears to be the major producer of IL-8, a proinflammatory cytokine with potent neutrophil chemotactic activity. By using Northern blot analysis, we have examined the kinetics of IL-8 mRNA expression in P. haemolytica LPS-stimulated bovine alveolar macrophages and found that 1 ng of LPS per ml induces maximal expression of IL-8 mRNA. The results also indicate a biphasic time course expression pattern in which IL-8 mRNA levels peak between 1 and 2 h in the first phase and between 16 and 24 h in the second phase (P < 0.01). In addition, monospecific polyclonal antibodies were used to demonstrate the role of tumor necrosis factor alpha (TNF-alpha) in the second phase of IL-8 mRNA expression. Our findings support a role for P. haemolytica LPS and TNF-alpha in the induction of IL-8 from bovine alveolar macrophages.