Interleukin-6 production by tumor necrosis factor and lipopolysaccharide-stimulated rat renal cells

Interleukin-6 (IL-6) is produced by various cell types, including monocytes, fibroblasts, and endothelial cells. IL-6 has also been detected in the urine of normal and renal transplant patients. Thus, the possible production of this cytokine by glomeruli and mesangial cells was investigated. Rat glomeruli were obtained by serial sieving of cortical homogenates of blood-free kidneys. Mesangial cells were obtained from the glomeruli and cultured under standard methods in RPMI 1640 medium containing 15% fetal calf serum. Glomeruli or confluent monolayers cells were then incubated in RPMI 1640 for 18 hr, in the presence or not of tumor necrosis factor-alpha (TNF alpha), lipopolysaccharide (LPS), or platelet-activating factor (PAF). IL-6 activity was measured using the IL-6-dependent cell line subclone (B 9-9) and expressed with respect to a standard curve established with recombinant IL-6. Glomeruli generate IL-6 upon TNF alpha (100 ng/ml) and LPS (1 microgram/ml), 11,500 +/- 3000 and 22,000 +/- 7500 U/ml, respectively. Nonstimulated mesangial cells produced 50 +/- 5 U/ml (mean +/- SEM, n = 4) of IL-6. TNF alpha (1 ng/ml) and LPS (1 microgram/ml) induced the production of 800 +/- 90 and 40,000 +/- 5000 U/ml, respectively (n = 4). In contrast, PAF (0.1 nM-1 microM) did not increase IL-6 production from glomeruli or mesangial cells. These results demonstrate that renal cells spontaneously generate minimal amounts of IL-6 and that this production is significantly increased by TNF alpha or LPS. A synergy between LPS and TNF alpha was induced in glomerular cells with 10 ng/ml of TNF alpha and graded concentrations of LPS. Thus, the production of IL-6 by glomerular cells and its modulation by other cytokines or endotoxins may play a role in the local immunological processes leading to immune glomerular diseases.     

Induction of tolerance in macrophages by cholera toxin B chain.

Model systems of human type 1 diabetes have revealed an important role of cellular immune reactions involving macrophages and T cells in the destruction of autologous insulin-producing pancreatic beta cells. Recently, the cholera toxin B chain (CTB) was found to suppress T cell-dependent autoimmune diseases including autoimmune diabetes of nonobese diabetic mice. Therefore, we tested the hypothesis that CTB exerts much of its immunomodulatory activity by targeting macrophages. These studies are reviewed here. Cells of the human monocyte line Mono Mac 6 were exposed to CTB and subsequently tested for proinflammatory immunoreactivity in response to challenge with endotoxin (LPS from Escherichia coli, 10 ng/ml for 5 h). Incubation of monocytes with CTB (10 microgram/ml) suppressed a later proinflammatory response to LPS as demonstrated by suppression of TNFalpha release from 6.7 +/- 0.7 ng/ml in cultures without CTB preexposure to 1.8 +/- 1.1 ng/ml in CTB-pretreated cells (p < 0.001). In contrast, the release of IL-10 remained inducible after CTB pretreatment. RT-PCR analysis showed that the suppression of TNFalpha production occurred at the level of mRNA formation. Control experiments excluded a role of possible contamination of CTB by endotoxin or the intact cholera toxin. Tolerance induction was maximal after 5 h of CTB exposure and persisted for 24 h. The suppressive effect of CTB was dose-dependent and no more recognizable at 相似文献   

Downregulation of tumor necrosis factor expression in the human Mono-Mac-6 cell line by lipopolysaccharide

Mono-Mac-6 cells, but not U937 cells, can be induced to rapidly express tumor necrosis factor (TNF) mRNA and protein when triggered with lipopolysaccharide (LPS) at 1 microgram/ml. Preincubation of the cells for 3 d with low amounts of LPS (10 ng/ml) results in nearly complete suppression of TNF secretion. This downregulation appears to occur at the pretranslational level since specific mRNA is virtually undetectable under these conditions. By contrast, the same preincubation with 10 ng/ml LPS results in enhanced phagocytosis (28.6-67.2% for Staphylococcus aureus), demonstrating that not all monocyte functions are suppressed. While these results show that only stringent exclusion of LPS from culture media allows for induction of TNF in the Mono-Mac-6 cell line, the pronounced effect of LPS preincubation may also provide a suitable model with which to study the mechanisms of LPS-induced desensitization.     

Cytokine induction by lipopolysaccharide (LPS) corresponds to lethal toxicity and is inhibited by nontoxic Rhodobacter capsulatus LPS.

Many pathological effects of gram-negative bacteria are produced by their cell wall-derived lipopolysaccharides (LPSs). Differing pathogenicity of gram-negative LPSs, however, may depend on their capacities to induce cytokines. Thus, we studied the lethal toxicity of four nonenterobacterial LPSs and compared it with their capacity to induce mononuclear cell (MNC)-derived interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). Unstimulated MNC did not release these cytokines. LPS from the phototrophic strain Rhodobacter capsulatus 37b4 elaborated little toxicity in galactosamine-treated mice (10 micrograms of LPS per mouse was the 100% lethal dose [LD100]) and induced IL-1 and IL-6 release only at high concentrations (10 to 50 micrograms of LPS per ml). R. capsulatus LPS failed to induce TNF activity even at the highest concentration tested (100 micrograms of LPS per ml). In contrast, LPS derived from Pseudomonas diminuta NCTC 8545 or the nodulating species Bradyrhizobium lupini DSM 30140 and Rhizobium meliloti 10406 expressed lethal toxicity (LD100, 1,000, 100, and 10 ng per mouse, respectively) and induced IL-1 or IL-6 (10 to 100, 10, and 1 ng of LPS per ml, respectively) at concentrations 1,000- to 10,000-fold lower than effective levels of R. capsulatus LPS. LPSs from P. diminuta, B. lupini, and R. meliloti also stimulated TNF production and release. MNC accumulated cell-associated IL-1 activities under circumstances in which released activity was readily detected. The cells contained only scant IL-6 activity, indicating release of this mediator rather than intracellular accumulation. Antisera to the respective cytokines inactivated biological activities of the samples selectively. The R. capsulatus LPS inhibited cytokine induction by LPS from P. diminuta, B. lupini, and R. meliloti in coincubation experiments. These results show that the in vivo lethality of the LPSs tested correlates with the induction of monocyte-derived cytokines in vitro. The results of this study suggest that the different lethality of various LPSs from gram-negative bacteria may be due to the differential ability of these LPSs to induce cytokine production.     

Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-1 or tumour necrosis factor.

Cultured normal human fibroblasts were stimulated to produce neutrophil-activating protein/interleukin-8 (IL-8) in response to IL-1 alpha (0.1-1000 U/ml) or tumour necrosis factor (TNF) alpha (0.1-1000 U/ml). Induction of mRNA for IL-8 in fibroblasts was rapid (within 30 min) and maximal responses were obtained with either 100 U/ml IL-1 alpha or 100 U/ml TNF alpha. Expression of mRNA for IL-8 was accompanied by the production of high levels of neutrophil chemotactic activity. IL-1 alpha (1000 U/ml), but not TNF alpha, induced mRNA for IL-8 in cultured normal human keratinocytes. The relevance of production of IL-8 by these cell types was evaluated further by comparing the local inflammatory effects of IL-1 alpha, TNF alpha and IL-8. Intradermal injection of either recombinant IL-8, IL-1 alpha or TNF alpha lead to a similar in vivo effect, i.e. dose-dependent accumulation of lymphocytes and polymorphonuclear leucocytes at sites of injection. The in vivo attraction of neutrophils and lymphocytes to the site of injection by TNF or IL-1 (which is not chemotactic for neutrophils or lymphocytes in vitro), may be partly mediated by locally produced IL-8. Thus, IL-8 may be a vital participant in the cascade of interacting cytokines that is induced by tissue injury and immunologically induced inflammation.     

Extracts of scabies mites (Sarcoptidae: Sarcoptes scabiei) modulate cytokine expression by human peripheral blood mononuclear cells and dendritic cells

We performed a series of experiments to determine if human peripheral blood mononuclear cells (PBMCs) from a healthy donor and dendritic cells (NHDCs) derived from these PBMCs reacted to molecules in a scabies extract. PBMCs extravasate from the circulatory system and enter tissues such as scabietic lesions, where monocytes become macrophages. Cells were cultured in medium alone or medium containing 50 microg/ml of Sarcoptes scabiei (SS) extract, 50 ng/ml E. coli lipopolysaccharide (LPS), or SS + LPS together. Supernatants were collected and assayed by enzyme-linked immunosorbent assay (ELISA) for specific cytokines. PBMCs stimulated with SS or LPS exhibited moderately upregulated production of interleukin (IL)-1beta and huge increases in secretions of IL-6, IL-8 and TNF-alpha. Cells co-stimulated with both SS and LPS generally secreted more of these cytokines than cells stimulated with either SS or LPS alone. LPS induced a small amount of IL-1alpha secretion, whereas SS did not, and neither additive resulted in the production of IL-10. NHDCs did not produce IL-1alpha, IL-1beta, IL-6, IL-8, or IL-10 in response to stimulation with SS. These cells did produce IL-6, IL-8, and tumor necrosis factor (TNF)-alpha in response to LPS. When cells were co-stimulated with both LPS and SS, the production of IL-6 and IL-8 was significantly reduced compared with the levels secreted after LPS stimulation alone. These studies show that molecules in a whole body extract of S. scabiei modulate the function of PBMCs (probably monocytes) and dendritic cells.     

Expression of interleukin-8 by lipopolysaccharide-stimulated bone marrow-derived mononuclear cells.

Interleukin-8 (IL-8) is a potent neutrophil chemoattractant, produced by a variety of immune and nonimmune cells in response to exogenous and host-derived inflammatory stimuli. We demonstrate here that a suspension of normal bone marrow mononuclear cells, consisting principally of myeloid precursors, produces IL-8 in response to stimulation with lipopolysaccharide (LPS). IL-8-specific mRNA is rapidly induced, being detected first 30 min after stimulation. IL-8 is detected by enzyme-linked immunosorbent assay within 2 h of stimulation, with steady a increase in its level through 72 h. Further studies demonstrated that LPS could serve as a primary stimulus for the expression of IL-8, since LPS challenge in the presence of cycloheximide resulted in superinduction of bone marrow mononuclear cell-derived IL-8 mRNA. These investigations suggest that the stimulatory effect of LPS is independent of other cytokines such as IL-1 beta. When compared with LPS, IL-1 beta proved to be a weak signal for the expression of IL-8 by bone marrow mononuclear cells. In a dose-response study, the maximum stimulatory concentration of IL-1 beta (300 pg/ml) resulted in the production of 500 pg of IL-8 per 10(6) cells, whereas 1 microgram of LPS resulted in the production of 5.5 ng/10(6) cells. Although IL-1 beta was not a particularly potent stimulus for IL-8 production by bone marrow mononuclear cells, peripheral blood mononuclear cells were highly susceptible to IL-1 beta challenge. In addition, the potential dependence of LPS-induced marrow-derived IL-8 production on the intermediate synthesis of IL-1 beta was further investigated. Results of studies assessing kinetics, addition of cycloheximide, and blocking with IL-1 beta neutralizing antibody were all consistent with the ability of LPS to directly induce bone marrow-derived IL-8 independently of IL-1 beta. These investigations demonstrate that bone marrow may be a significant source of IL-8 and may play a significant role in acute infectious, inflammatory responses.     

Low Endotoxic Potential of Legionella pneumophila Lipopolysaccharide due to Failure of Interaction with the Monocyte Lipopolysaccharide Receptor CD14

Legionella pneumophila, a gram-negative bacterium causing Legionnaires’ disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.     

The Effect of Cytokines on Expression of Glutamic Acid Decarboxylase-65 in Cultured Islets

Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by lymphocytic infiltration of the pancreatic islets (insulitis). Cytokines released as part of the insulitis process have been suggested to play an important role in the beta cell lesion of IDDM. A possible diabetogenic effect of cytokines may be mediated by their inducing abnormal expression of islet cell autoantigens. Since glutamic acid decarboxylase-65 (GAD-65) is a target autoantigen in IDDM, we investigated whether the cytokines IL-1β, TNFα IFNγ altered islet cell expression of GAD-65 and whether the effect of cytokines on GAD-65 expression was similar to their effect on insulin secretion.

We found that: 1) IL-1β at low dose (1 U/ml) which stimulated insulin secretion, had no effect on GAD-65 expression, whereas higher doses of IL-1β (10, 100, 1000 U/ml) which inhibited insulin secretion, decreased GAD-65 expression. 2) TNFα at doses of 10, 100, 1000 U/ml which stimulated insulin secretion had no effect on GAD-65 expression. 3) IFNγ at doses of 10, 100, 1000 U/ml had no effect on insulin secretion or on GAD-65 expression. 4) In combination, IL-1β plus TNFα and IFNγ showed a similar inhibitory effect on GAD-65 expression as IL-1β alone.

In summary: 1) IL-1β dramatically inhibits GAD-65 expression. 2) TNFα and IFNγ have no effect on GAD-65 expression. Of these three cytokines, IL-1β is the primary cytokine affecting GAD-65 expression.     

Detection of Tumour Necrosis Factor from Lipopolysaccharide-Stimulated Human Mononuclear Cells by Enzyme-Linked Immunosorbent Assay and Cytotoxicity Bioassay

Tumour necrosis factor (TNF) or cachectin is an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF (rTNF)/100 microliter. There was no interference of medium, serum, plasma, spinal fluid, or urine and no cross-reaction with natural or recombinant IL-1-alpha, IL-1-beta, IL-2, IFN-gamma, or lymphotoxin (TNF-beta). Recovery of TNF added to the media was 85-123% (n = 22). The relative standard deviations within and between assays were 7% and 8%, respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30 microliter or 12.5 pg/30 microliter of rTNF. IL-1-alpha and IL-1-beta slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50-300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100-1000 ng/ml). In contrast, using 0.01-100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine. Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.     

Tumor necrosis factor induces enhanced responses to platelet-activating factor and differentiation in human monocytic Mono Mac 6 cells

Human Mono Mac 6 cells exhibit characterstics of mature blood monocytes. Treatment of these cells with human recombinant human tumor necrosis factor (TNF)resulted in an increase in phagocytosis and phorbol myristate acetate stimulated superoxide anion production at 12 h and growth retardation occurring at 24 h. Moreover, TNF induced a moderate increase of CD14 surface antigen expression, used as a phenotypic marker of monocyte/macrophage differentiation. Platelet-activating factor (PAF) stimulated a rapid rise in cytosolic free Ca⁺⁺ ([Ca⁺⁺]j) of 308 & 93 nM inTNF-treated cells compared to untreated cells (33 ± 8 nM, n = 4). The effect of TNF was dose and time dependent, evident after 12 h and maximal at 48 h. The enhanced PAF-induced [Ca⁺⁺]_i rise was inhibited by the PAF receptor antagonist L-659,989 and EGTA, indicating receptor-dependent Ca⁺⁺ influx. Furthermore, L-659,989 and PAF inhibited specific ³H-labeled PAF binding inTNF-treated, but not in untreated cells. Consistently, PAF stimulated arachidonic acid release only in TNF-treated cells. Preincubation of cells with anti-TNF monoclonal antibodies abolished TNF-induced effects, but failed to block lipopolysaccharide (LPS) effects. Distinct mechanisms of action by LPS were reflected by the different ability to induce surface antigen expression. In conclusion, the enhancement of PAF responses by TNF, associated with functional characteristics of differentiation in Mono Mac 6 cells, may represent a specific mechanism of cooperative interaction between PAF and TNF in inflammation, sepsis, immunoregulation and atherogenesis.     

Inhibition of lipopolysaccharide-induced in vitro desensitization by interferon-gamma

Exposure of Mono-Mac-6 cells to lipopolysaccharide (LPS) can induce rapid and transient expression of cytokines like tumor necrosis factor (TNF), interleukin 1 and interleukin 6. Preculture of Mono-Mac-6 cells in culture medium containing small amounts (1-50 ng/ml) of LPS for 3 days leads to an unresponsiveness to a subsequent stimulation with a high amount of LPS. This in vitro desensitization of a monocytic cell line may serve as a model for desensitization to LPS seen in vivo, for example in mice or man repetitively treated with LPS. Addition of interferon-gamma (IFN-gamma) to the Mono-Mac-6 cells during the LPS preculture period leads to an inhibition of desensitization, whereas addition of IFN-alpha or IFN-beta is not able to inhibit the LPS-induced desensitization. The inhibition of desensitization by IFN-gamma was dose dependent and time dependent. Preculture of Mono-Mac-6 cells with LPS leads to a strong reduction of TNF mRNA. This reduction of specific mRNA is also overcome by addition of IFN-gamma, but not by IFN-alpha and IFN-beta, indicating that pretranslational mechanisms are responsible for the regulation of TNF in desensitization.     

Production of tumor necrosis factor-alpha and interleukin-6 by human alveolar macrophages exposed in vitro to coal mine dust.

Following our previous demonstration of cytokine secretion by alveolar macrophages (AM) from coal miners and from patients with coal workers' pneumoconiosis, we investigated the effect of in vitro exposure to coal dust and to its silica content on tumor necrosis factor-alpha (TNF), interleukin (IL)-1 beta, and IL-6 production by normal human AM. TNF and IL-1 beta concentrations were estimated by a specific radioimmunoassay, while IL-6 levels were evaluated by the proliferation of 7TD1 cells. After 24-h culture, coal dust triggered a significant release of TNF and IL-6 at the dose of 0.1 mg/ml and more obviously at 1 mg/ml in comparison with titanium dioxide (TiO2), used as a biologically inert control dust (with 1 mg/ml of dust: 3,526 +/- 3,509 versus 330 +/- 138 pg TNF/ml and 224 +/- 74 versus 72 +/- 34 U IL-6/ml, respectively; P less than 0.01 in both cases). After 3-h culture, a significant TNF secretion as well as an increased TNF mRNA expression were also detected for AM stimulated by coal dust at variance with TiO2. In contrast, no modification of IL-1 beta concentration could be evidenced in AM exposed to coal dust, although we detected an increased expression of specific mRNA expression. In order to define the role of silica among the main components of coal dust in AM activation, we evaluated the effect of silica (alpha-quartz, 30 micrograms/ml, which is the concentration and the type of silica present in our coal dust) alone or mixed with TiO2 (1 mg/ml) on monokine production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of interleukin-1-β and tumor necrosis factor-α on cartilage proteoglycan metabolismin vitro

The activities of recombinant interleukin-1- (IL-1) and recombinant tumor necrosis factor- (TNF) on cartilage proteoglycan metabolism were compared in an organ culture system. IL-1, 1 to 100 ng/ml, and TNF, 10 to 1,000 ng/ml, increased proteoglycan degradation. The concentration-response curves were parallel. The timecourse for degradation was similar for the two cytokines during a 6 day incubation. Both cytokines inhibited the synthesis of new proteoglycan as measured by³⁵S incorporation. The inhibition curves were parallel and concentration-related between 1 and 10 ng/ml for IL-1 and between 10 and 100 ng/ml for TNF. Maximal inhibition was 60% in the presence of IL-1 (10 ng/ml) or TNF (100 ng/ml), and plateaued at higher concentrations. IL-1 was ten fold more potent than TNF in stimulating proteoglycan breakdown and inhibiting proteoglycan synthesis. Degradation in response to TNF, but not to IL-1, could be blocked by a polyclonal antibody to TNF. A polyclonal antibody to IL-1 could block proteoglycan breakdown in response to both cytokines suggesting that TNF may be mediating proteoglycan degradation by inducing the production of interleukin-1.     

Effect of interferon-gamma,tumor necrosis factor,interleukin-1 and interleukin-6 on the modulation of anti-tumor responses of bone marrow derived macrophages

Non-adherent bone marrow cells (NABMC) obtained from BALB/c mice were incubated in medium alone or containing granulocyte — macrophage-colony stimulating factor(GM-CSF) or macrophage-colony stimulating factor (M-CSF) for 4 days to obtain bone marrow derived macrophages. Treatment of GM-CSF or M-CSF derived macrophages with interferon-gamma (IFN-γ) (50 U/ml), tumor necrosis factor (TNF) (500 U/ml), interleukin-1 (IL-1) (200 U/ml) or interleukin-6 (IL-6) (100 U/ml) for 24 h rendered them significantly cytotoxic to different tumor cells. These macrophages also produced enhanced amounts of soluble or membrane associated TNF. Medium derived macrophages showed little cytotoxicity against tumor cells and production of TNF on treatment with TNF, IFN-γ, IL-1 or IL-6. M-CSF or GM-CSF derived macrophages on treatment with IFN-γ showed enhanced release of nitrite as compared to medium derived macrophages. TNF, IL-1 or IL-6 did not induce nitrite production in bone marrow derived macrophages. Out of the different combinations tested, only IFN-γ plus TNF-treated macrophages showed enhancement in nitrite production as compared to that of IFN-γ alone.     

Purified Shiga-like toxins induce expression of proinflammatory cytokines from murine peritoneal macrophages.

Infections with Shiga toxin-producing Shigella dysenteriae type 1 and Shiga-like toxin (SLT)-producing Escherichia coli cause outbreaks of bloody diarrhea in which patients are at risk for developing life-threatening complications involving the renal and central nervous systems. Histopathology studies and in vitro experiments suggested that the toxins damage toxin receptor-expressing endothelial cells (EC) lining glomerular and central nervous system capillaries. In the presence of inducible host factors (cytokines), EC sensitivity to SLT toxicity was increased approximately 1 million-fold. We hypothesized that to manifest the vascular lesions characteristic of infection with toxin-producing bacteria, two signals were needed: systemic toxins and elevated proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 1 [IL-1], and IL-6). Human EC do not secrete these cytokines when stimulated with SLTs in vitro, suggesting that additional cells may be involved in pathogenesis. Therefore, we carried out comparative analyses of the capacity of purified (endotoxin-free) SLTs and lipopolysaccharides (LPS) to induce cytokine mRNA and proteins from murine macrophages. The cells were essentially refractory to SLT cytotoxicity, expressing low to undetectable levels of toxin receptor. SLTs and LPS induced TNF activity and IL-6 expression from macrophages, although dose response and kinetics of cytokine induction differed. LPS was a more effective inducing agent than SLTs. SLT-I-induced TNF activity and IL-6 expression were delayed compared with induction mediated by LPS. IL-1 alpha production required approximately 24 h of exposure to SLTs or LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice produced low levels of TNF activity when treated with SLT-I, suggesting that LPS and SLTs may utilize separate signaling pathways for cytokine induction.     

IFN-gamma induced persistent Chlamydia pneumoniae infection in HL and Mono Mac 6 cells: characterization by real-time quantitative PCR and culture

Growth of Chlamydia pneumoniae during gamma interferon (IFN-gamma) induced persistent infection in epithelial (HL) and monocyte-macrophage (Mono Mac 6) cell lines was studied by a quantitative real-time PCR and passage. When HL cultures were treated with IFN-gamma (25 U/ml), the replication of C. pneumoniae DNA was unaffected while differentiation into infectious elementary bodies (EB) was strongly inhibited, and in contrast to the untreated cultures, no second cycle of infection was observed. The estimated doubling time of C. pneumoniae genomes was 6-7 h in both IFN-gamma treated and untreated HL cultures. At 72 h post inoculation, most infectious EBs were released from untreated cultures, whereas in IFN-gamma treated HL cells >90% of C. pneumoniae genomes were in non-infectious form. A higher dose (1000 U/ml) of IFN-gamma was needed to restrict growth of C. pneumoniae in Mono Mac 6 cells. In untreated Mono Mac 6 cultures, the growth curve of C. pneumoniae resembled that observed in HL cells, except that no second cycle of infection could be detected. In IFN-gamma treated Mono Mac 6 cultures, the number of infectious C. pneumoniae EBs recovered decreased gradually after 3 days post inoculation, while C. pneumoniae genome load remained unaltered suggesting persistence of C. pneumoniae also in these cells.     

Immunobiological activities of Helicobacter pylori porins.

Studies were carried out on some biological activities of Helicobacter pylori porins in vitro. We extracted and purified a porin with an apparent molecular mass of 30 kDa. Human polymorphonuclear leukocytes preincubated with H. pylori porins showed a decrease of chemotaxis, of adherence to nylon wool, and of chemiluminescence. Used as chemotaxins in place of zymosan-activated serum or as chemotaxinogens in place of zymosan, the porins induced polymorphonuclear leukocyte migration. Human monocytes and lymphocytes cultivated in the presence of H. pylori porins released cytokines. Release of the various cytokines studied was obtained with differentiated kinetics and at various porin concentrations. Starting only 3 h after culture, tumor necrosis factor alpha is released quickly, reaching a peak at 18 h, at a porin concentration of 1 microgram/ml/10(6) cells. Interleukin-6 (IL-6) appears later, with a peak at 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells, while IL-8 is released after 6 h of culture, with a peak at 24 h, at a porin concentration of 10 micrograms/ml/10(6) cells. Lymphocytes stimulated by H. pylori porins release gamma interferon after 18 h of culture at higher concentrations of porins (20 micrograms/ml/10(6) cells). Granulocyte macrophage colony-stimulating factor is released from 6 to 48 h at a concentration of 1 microgram/ml/10(6) cells, while both IL-3 and IL-4 are released after 18 h of culture at different porin concentrations (0.1 and 1 microgram/ml/10(6) cells, respectively). Our results lead us to think that during H. pylori infection, surface components, porins in particular, are able to induce a series of chain reactions ranging from the inflammatory to the immunological responses.     

The involvement of protein kinase C, calcium, and 5-lipoxygenase in the production of tumor necrosis factor by a cloned interleukin-3 dependent cell line with natural cytotoxic activity

The cloned interleukin-3 dependent cell line, M1-A5 was studied to determine whether protein kinase C, calcium mobilization, and 5-lipoxygenase activity were involved in the signal transduction pathways required for the production of TNF. TNF release was stimulated by 10 ng/ml phorbol myristate acetate (PMA), 2 microM calcium ionophore A23187, and 1 microgram/ml lipopolysaccharide (LPS) with synergism seen between PMA and A23187. All signals were blocked by phloretin and the PMA signal was blocked by H-7, both drugs acting as protein kinase C inhibitors. Desensitization of protein kinase C by PMA (1 microgram/ml for 24 h) provided evidence that both PMA- and LPS-stimulated TNF production were protein kinase C-dependent while A23187-stimulated TNF production was not. Both the calcium chelator, EGTA, and the intracellular calcium antagonist, TMB-8, inhibited TNF production stimulated by all agents, indicating that TNF stimulation by all agents was calcium dependent. Finally, the 5-lipoxygenase inhibitors, ketoconazole and L-656,224, but not the cyclo-oxygenase inhibitor ASA, inhibited TNF stimulated by all agents. These findings indicate that, although TNF production by M1-A5 cells can be stimulated either by a calcium/protein kinase C- or by a calcium-dependent signal, there is a convergence of signals at the level of 5-lipoxygenase activation.