Binding and biological properties of lipopolysaccharide Proteus vulgaris O25 (48/57)–chitosan complexes

In this study the negatively charged Proteus vulgaris O25 LPS was chosen for studying interaction with polycationic chitosan. The complex formation of LPS with chitosan was demonstrated using gradient centrifugation and laser interferometry method. The presented results have shown that laser interferometry method is sensitive enough for LPS–chitosan interaction studies. The changing in the ultra structure of LPS during binding with chitosan was observed by electronic microscope. The interaction of P. vulgaris O25 LPS with chitosan was shown to modulate significantly the biological activities of LPS. The toxicity of P. vulgaris O25 LPS decreased 10-fold after forming complexes with chitosan at injection to mice in the similar concentration of endotoxin. The complex LPS–chitosan was less effective than LPS alone in Limulus amabocyte lysate assay. Induction of TNF biosynthesis by LPS–chitosan complex was found to be 65% lower than that by parent LPS at concentration of 100 ng/ml.     

The effect of temperature on the interaction of Yersinia pseudotuberculosis lipopolysaccharide with chitosan

The mechanism of binding of lipopolysaccharide (LPS) from Yersinia pseudotuberculosis to low-molecular-weight chitosan was investigated using sedimentation analysis, centrifugation in glycerol and percoll density gradients, and isopicnic centrifugation in cesium chloride. The LPS interaction with chitosan was shown to be a multistage process that depended on time and reaction temperature. A stable LPS-chitosan complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). This temperature caused the LPS dissociation and promoted its binding to chitosan. The LPS binding to chitosan results in further dissociation of the endotoxin and formation of the complex with a molecular weight that is tens of times less than the initial molecular weight of LPS. The obtained complex remained stable in solutions of high ionic strength.     

Comparative study of electrokinetic potentials and binding affinity of lipopolysaccharides-chitosan complexes

Electrokinetic properties of complexes of chitosan (Ch) with lipopolysaccharides (LPSs) from Escherichia coli O55:B5, Yersinia pseudotuberculosis 1B 598, and Proteus vulgaris O25 (48/57) and their size distribution were investigated using zeta-potential distribution assay and quasi-elastic light scattering. The interaction of LPS from different microorganisms with chitosan at the same w/w ratio of components (1:1) resulted in the formation of complexes in which the negative charge of LPS was neutralized (LPS from E. coli) or overcompensated (Y. pseudotuberculosis and P. vulgaris). The changing in size of the endotoxin aggregates during binding with chitosan was observed. The binding constants of chitosan with LPSs were determined by a method with using the anionic dye Orange II. The LPS from E. coli possess higher affinity to chitosan in comparison with the two others samples of endotoxin.     

Selection of novel analogs of thalidomide with enhanced tumor necrosis factor alpha inhibitory activity.

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) is thought to mediate both protective and detrimental manifestations of the inflammatory response. Recently, thalidomide (alpha-N-phthalimidoglutarimide) was shown to partially inhibit monocyte TNF alpha production (by 50-70%) both in vivo and in vitro. More efficient inhibition of TNF alpha may, however, be necessary to rescue the host from more acute and extensive toxicities of TNF alpha-mediated inflammation. MATERIALS AND METHODS: Three structural analogues of thalidomide were selected for study based on increased activity against TNF alpha production. The parent drug and the analogs were tested in vitro in human peripheral blood mononuclear cell cultures for their effects on lipopolysaccharide (LPS) induced cytokine protein and mRNA production using ELISAs and Northern blot hybridization. The in vitro effects of the drugs were then confirmed in vivo in a mouse model of LPS induced lethality. RESULTS: The new compounds (two esters and one amide) showed increased inhibition of TNF alpha production by LPS-stimulated human monocytes, relative to the parent drug thalidomide. The analogs and the parent drug enhanced the production of interleukin 10 (IL-10), but had little effect on IL-6 and IL-1 beta protein and mRNA production. When tested in vivo, the amide analog protected 80% of LPS-treated mice against death from endotoxin induced shock. CONCLUSIONS: Analogs of thalidomide designed to better inhibit TNF alpha production in vitro have correspondingly greater efficacy in vivo. These finding may have therapeutic implication for the treatment of human diseases characterized by acute and extensive TNF alpha production such as tuberculous meningitis or toxic shock.     

Modulation of lipopolysaccharide-induced production of tumor necrosis factor, interleukin 1, and interleukin 6 by synthetic precursor Ia of lipid A

Abstract Endotoxin (lipopolysaccharide, LPS) induces the production of mediators of inflammation, which exerts pathophysiological effects such as fever or shock in mammals. In the present study we have investigated the modulation of LPS by the synthetic non-active tetraacylated precursor Ia of lipid A (compound 406) in the induction of tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 (IL-6) in human peripheral blood mononuclear cells (PBMC) and in human peripheral blood monocytes (PBMo). PBMC stimulated with LPS released TNF in a concentration dependent manner. Release of biologically active TNF, IL-1 and IL-6 was first detectable 4 h after LPS stimulation. Compound 406 alone in all concentrations tested did not induce TNF, IL-1 or IL-6 release, intracellular TNF or IL-1β, or mRNA for TNF or IL-1. Added to PBMC 1 h before LPS compound 406 enhanced or suppressed TNF release and suppressed IL-1 and IL-6 release depending on the ratio of concentrations between stimulator (LPS) and modulator (compound 406). In contrast to LPS stimulation alone TNF, IL-1 and IL-6 release in presence of compound 406 was delayed and first detectable after 6 to 8 h. Compound 406 was able to suppress LPS-induced intracellular TNF and IL-1β in PBMC. Added to PBMo 1 h before LPS it totally inhibited the production of mRNA for TNF and IL-1. When added to PBMC 1 h after LPS, TNF release was suppressed in a concentration-dependent way and release of biologically active TNF, IL-1 and IL-6 could again be detected for the first time after 4 h. Compound 406 was not able to inhibit phorbol 12-myristate 13-acetate (PMA)-induced TNF and IL-1 release in PBMo which suggests that its modulating effect is LPS-specific. This study provides evidence that the modulating effect of compound 406 on the LPS induction of TNF, IL-, 1 and IL-6 could be due to competitive binding.     

Interaction of chitosans and their N-acylated derivatives with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the natural polycation chitosan and its derivatives--high molecular weight chitosans (80 kD) with different degree of acetylation, low molecular weight chitosan (15 kD), acylated oligochitosan (5.5 kD) and chitooligosaccharides (biose, triose, and tetraose)--were studied using ligand-enzyme solid-phase assay. The LPS-binding activity of chitosans (80 kD) decreased with increase in acetylation degree. Affinity of LPS interaction with chitosans increased after introduction of a fatty acid residue at the reducing end of chitosan. Activity of N-monoacylated chitooligosaccharides decreased in the order: oligochitosan --> tetra- > tri- --> disaccharides. The three-dimensional structures of complexes of R-LPS and chitosans with different degree of acetylation, chitooligosaccharides, and their N-monoacylated derivatives were generated by molecular modeling. The number of bonds stabilizing the complexes and the energy of LPS binding with chitosans decreased with increase in acetate group content in chitosans and resulted in changing of binding sites. It was shown that binding sites of chitooligosaccharides on R-LPS overlapped and chitooligosaccharide binding energies increased with increase in number of monosaccharide residues in chitosan molecules. The input of the hydrophobic fragment in complex formation energy is most prominent for complexes in water phase and is due to the hydrophobic interaction of chitooligosaccharide acyl fragment with fatty acid residues of LPS.     

Determination of binding constants of lipopolysaccharides of different structure with chitosan

The interaction of endotoxins--lipopolysaccharides (LPS) different in degree of the O-specific chain polymerization--with 20- and 130-kD chitosan was studied using the competitive binding of LPS with the complex of chitosan-anionic dye (tropaeolin 000-2) and the direct binding of (125)I-labeled LPS with chitosan immobilized on Sepharose 4B. The interaction of 20-kD chitosan with LPS was non-cooperative, and immobilization of the polycation on Sepharose resulted in its binding to (125)I-labeled LPS with a positive cooperativity. The interaction of LPS possessing a long O-specific chain with 130-kD chitosan was characterized by negative cooperativity. Binding constants of LPS with the polycation and the number of binding sites per amino group of chitosan were determined. The interaction affinity and stoichiometry of the LPS-chitosan complexes significantly depend on the LPS structure and concentration in the reaction mixture. The increase in the length of carbohydrate chains of LPS results in increase in the binding constants and decrease in the bound endotoxin amount.     

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-beta was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-kappaB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.     

Role of interleukin-1 and tumour necrosis factor in leukocyte recruitment to acute dermal inflammation

The cytokines IL-1 and TNF-alpha are involved in inflammation and their production is stimulated by various agents, especially endotoxin (LPS). Here, using the human IL-1 receptor antagonist (IL-1RA) and a new monoclonal antibody (mAb 7F11) to rabbit TNF, the role of endogenous IL-l and TNF production in acute (3h) leukocyte (PMNL) recruitment to dermal inflammation in rabbits has been studied. IL-1RA inhibited by 27% the PMNL accumulation in reactions induced by killed Escherichia coli (p < 0.05) but not by LPS. The monoclonal antibody to TNF inhibited by 27% and 38% (p < 0.002) the PMNL accumulation in LPS and E. coli reactions respectively, but a combination of the mAb with IL-1RA was not more effective. Treatment of human umbilical vein endothelium with LPS for 3 h activated endothelium to induce PMNL transendothelial migration in vitro, which was not inhibited by IL-1RA, antibody to TNF-alpha, IL-1 or to IL-8. In conclusion, TNF and IL-1 may partially mediate acute PMNL infiltration in vivo to LPS and Gram negative bacteria, but there is a major IL-1/TNF independent mechanism, at least in dermal inflammation, which may be due to direct LPS activation of the microvasculature or perhaps the generation of cytokines other than IL-1 and TNF.     

Interaction of N-acylated and N-alkylated chitosans included in liposomes with lipopolysaccharide of gram-negative bacteria

The interactions of lipopolysaccharide (LPS) with the polycation chitosan and its derivatives — high molecular weight chitosans (300 kDa) with different degree of N-alkylation, its quaternized derivatives, N-monoacylated low molecular weight chitosans (5.5 kDa) — entrapped in anionic liposomes were studied. It was found that the addition of chitosans changes the surface potential and size of negatively charged liposomes, the magnitudes of which depend on the chitosan concentration. Acylated low molecular weight chitosan interacts with liposomes most effectively. The binding of alkylated high molecular weight chitosan with liposomes increases with the degree of its alkylation. The analysis of interaction of LPS with chitoliposomes has shown that LPS-binding activity decreased in the following order: liposomes coated with a hydrophobic chitosan derivatives > coated with chitosan > free liposomes. Liposomes with N-acylated low molecular weight chitosan bind LPS more effectively than liposomes coated with N-alkylated high molecular weight chitosans. The increase in positive charge on the molecules of N-alkylated high molecular weight chitosans at the cost of quaternization does not lead to useful increase in efficiency of binding chitosan with LPS. It was found that increase in LPS concentration leads to a change in surface ζ-potential of liposomes, an increase in average hydrodynamic diameter, and polydispersity of liposomes coated with N-acylated low molecular weight chitosan. The affinity of the interaction of LPS with a liposomal form of N-acylated chitosan increases in comparison with free liposomes. Computer simulation showed that the modification of the lipid bilayer of liposomes with N-acylated low molecular weight chitosan increases the binding of lipopolysaccharide without an O-specific polysaccharide with liposomes due to the formation of additional hydrogen and ionic bonds between the molecules of chitosan and LPS.     

Biphasic production of IL-8 in lipopolysaccharide (LPS)-stimulated human whole blood. Separation of LPS- and cytokine-stimulated components using anti-tumor necrosis factor and anti-IL-1 antibodies.

TNF, IL-1, and IL-6 are integral components of the cytokine cascade released in the response to inflammatory stimuli such as LPS. IL-8 is produced both in response to LPS as well as TNF and IL-1. The early, local production of TNF and IL-1 may therefore contribute to the subsequent expression of IL-8. This hypothesis was tested using LPS-stimulated human whole blood as an ex vivo model of local cytokine production. The production of TNF, IL-1 alpha, IL-1 beta, IL-6, and IL-8 was found to be responsive to a wide range of LPS concentrations (0.1 ng/ml-10 micrograms/ml). These cytokines were first detected between 1 to 4 h post-LPS stimulation, and reached plateau levels after 6 to 12 h. IL-8, however, also displayed a secondary wave of production, with the levels again increasing between 12 to 24 h. The IL-8 present in the plasma after LPS stimulation was biologically active, as assessed by neutrophil chemotaxis. In further studies, addition of anti-TNF and anti-IL-1 neutralizing antibodies, alone and in combination, to LPS-stimulated blood resulted in nearly complete ablation of the secondary phase of IL-8 synthesis at both the levels of protein and mRNA, while leaving the first, LPS-mediated phase of IL-8 synthesis unaffected. This model of cytokine production in human whole blood may reflect the sequence of events in a localized environment of inflammation where both a primary stimulus and the induced early cytokine mediators may serve to elicit multiple, temporally distinct phases of IL-8 production.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.     

Modification of immunopharmacological activities of synthetic monosaccharide lipid A analogue, GLA60, by lysozyme.

Recent studies by our group suggested that lysozyme has a high affinity for bacterial lipopolysaccharide (LPS) of both the smooth and rough forms, and inhibits various immunomodulatory activities of LPS. GLA60 is a synthetic monosaccharide analogue of bacterial lipid A well known as having most of the activities of lipid A with very low toxicity. In this study, we characterized the interaction of lysozyme with GLA60 in comparison to that with Escherichia coli 0111 LPS (smooth form) by means of an immunopharmacological approach. Using dansylated lysozyme (DNS-LZM) as a probe, LZM was found to bind to GLA60. The mitogenic and polyclonal B-cell activating activities were significantly reduced by complex formation. However, there was no inhibitory effect on GLA60 induced production of IL-1 and TNF of macrophages. Interestingly, the activities of macrophages induced by the complex were found to be significantly higher than those induced by GLA60 itself. In contrast, the activities of 0111 LPS were significantly inhibited by LZM. Since the GLA60-LZM complex produced a turbid suspension but the 0111 LPS-LZM complex remained soluble, we consider that the activities of GLA60 alone were mediated by the common functional LPS receptor for dispersed form in both macrophages and B-lymphocytes, but activation of macrophages by the complex was mediated either by another LPS receptor not present in B-lymphocytes or through the phagocytic function of macrophages.     

Monokine production by hypersensitivity (Schistosoma mansoni egg) and foreign body (Sephadex bead)-type granuloma macrophages. Evidence for sequential production of IL-1 and tumor necrosis factor

The present study examined the potential role of IL-1 and TNF in granuloma formation. Mice were given i.v. injections of Schistosoma mansoni eggs or Sephadex beads to induce synchronous immune T cell-mediated (hypersensitivity type) or nonimmune (foreign-body type) granulomas, respectively. Granuloma macrophages isolated at 2, 4, 8, 16, and 32 days of granuloma formation were evaluated for their capacity to produce IL-1 and TNF in response to 1 microgram/ml LPS. This was related to circulating levels of the acute phase protein, serum amyloid P (SAP) and expression of Ia Ag by monocytes and macrophages. Macrophages from nonimmune bead lesions were generally weak producers of IL-1 and TNF. In contrast, those from T cell-mediated egg lesions produced significant levels of both monokines. Moreover, there was a clear pattern of sequential monokine production such that IL-1 was produced in greatest amounts early (2 to 4 days), whereas TNF was produced later (8 to 16 days). Levels of SAP showed an initial sharp rise following particle embolization, then decreased rapidly in bead injected animals. However, mice with immune granulomas showed a prolonged elevation in SAP levels that corresponded to the period of maximal IL-1 production (2 to 4 days). Macrophage/monocyte Ia Ag expression was greatest at 8 to 16 days, corresponding to the period of TNF production. Bead injected animals showed low levels of Ia expression over the study period. These findings suggest that IL-1 may be important in the early recruitment stages of granuloma formation while TNF may take part in later maintenance or effector functions. The extent of production of both is likely influenced by the local or systemic milieu of lymphokines.     

Regulation of lipopolysaccharide-induced tumor necrosis factor production by cyclosporin A in mice primed with muramyl dipeptide

Abstract The effect of cyclosporin A (CsA) on tumor necrosis factor (TNF) or interleukin-6 (IL-6) production was evaluated in vivo in primed or unprimed mice challenged with lipopolysaccharide (LPS). Both pretreatment with BCG infection or with muramyl dipeptide (MDP) prior to LPS challenge resulted in an increase in the cytokine bioactivity level in the blood. CsA administration inhibited the TNF production. In unprimed mice, either normal or sensitized to LPS lethality by galactosamine treatment, a marked decrease in the cytokine level was observed after injection of CsA. After adrenalectomy, the yield of both TNF and IL-6 following LPS injection was markedly elevated but decreased by CsA administration. Ex vivo experiments have shown that the inhibitory effect of CsA could be demonstrated at the level of macrophages from mice previously given the drug. If mice had received MDP, in vitro responses of cells to LPS were enhanced but again CsA decreased the mRNA expression and protein secretion.     

Interleukin 8 production in whole blood assays: Is interleukin 10 responsible for the downregulation observed in sepsis?

Reduced cytokine production in ex vivo cultures has been regularly reported in patients suffering from sepsis syndrome. Using whole blood assays, we have now demonstrated that in sepsis patients, normal production of IL-8 was achieved with the higher concentration of lipopolysaccharide (LPS; 1 microg/ml) and with heat-killed streptococci, whereas the IL-8 production induced by lower LPS concentration (0.1 microg/ml) was significantly reduced as compared to healthy controls. In contrast, in patients undergoing cardiac surgery associated with cardio-pulmonary bypass, a group of patients with inflammation in the absence of infectious insult, none of the studied IL-8 productions were affected. Among the various anti-inflammatory cytokines known to regulate IL-8 production which we tested (i.e. IL-4, IL-10, IL-13, TGF-beta), IL-10 was the most active inhibitory cytokine in whole blood assays performed with blood samples from healthy subjects. However, its activity was not influenced by the amounts of LPS used. In addition, IL-10 also inhibited the heat-killed streptococci-induced IL-8 production and was the only cytokine to inhibit the release of IL-8 when TNF was added to LPS. It is worth noting that IL-13 which also inhibited the heat-killed streptococci-induced IL-8 production, failed to do so when the TNF production was analysed. Together, these data suggest that while circulating IL-10 in septic patients may be responsible for the hyporeactivity of circulating leukocytes, its presence is not sufficient to explain the observed dysregulation which occurs in septic patients.     

Neutrophil leukocyte emigration induced by endotoxin. Mediator roles of interleukin 1 and tumor necrosis factor alpha 1

The hypothesis that cytokines mediate neutrophil emigration induced by endotoxin (LPS) was studied by examining the potency, the kinetics of neutrophil emigration, and the tachyphylaxis of intradermal sites with IL-1, TNF-alpha and LPS. Human rIL-1 alpha and IL-1 beta, synthetic lipid A, and LPS were several orders of magnitude more potent than human rTNF. The kinetic profiles of neutrophil emigration induced by IL-1 alpha, TNF, and LPS were characterized by minimal emigration in the first 30 min, followed by rapid and transient emigration. After the injection of LPS, the onset and the time at which the rate of emigration was maximal consistently appeared 30 min later than IL-alpha or TNF, suggesting that neutrophil emigration in response to LPS was mediated by a locally generated cytokine. IL-1 and TNF were then examined as potential secondary mediators of LPS-induced emigration by comparing the patterns of tachyphylaxis between LPS and IL-1 alpha or TNF; i.e., the magnitude of neutrophil emigration into inflammatory sites was compared with sites injected 6 h previously (desensitizing injections) with a cytokine or with LPS. Tachyphylaxis was dose dependent with each and also between the IL-1 species; therefore, when tachyphylaxis between the cytokines and LPS was examined, relatively higher doses were selected for the desensitizing injections than for the test injections. With this approach, desensitizing injections of IL-1 alpha diminished the neutrophil accumulation after LPS, and LPS also desensitized sites to IL-1 alpha. However, tachyphylaxis was not observed between TNF and LPS, or between TNF and IL-1 alpha. These data suggest that IL-1, but not TNF, is a potential mediator of LPS-induced neutrophil emigration.     

Activation of individual tumor necrosis factor receptors differentially affects stem cell growth factor and cytokine production

Necrotizing enterocolitis (NEC) is an emergency of the newborn that often requires surgery. Growth factors from stem cells may aid in decreasing intestinal damage while also promoting restitution. We hypothesized that 1) TNF, LPS, or hypoxia would alter bone marrow mesenchymal stem cell (BMSC) TNF, IGF-1, IL-6, and VEGF production, and 2) TNF receptor type 1 (TNFR1) or type 2 (TNFR2) ablation would result in changes to the patterns of cytokines and growth factors produced. BMSCs were harvested from female wild-type (WT), TNFR1 knockout (KO), and TNFR2KO mice. Cells were stimulated with TNF, LPS, or hypoxia. After 24 h, cell supernatants were assayed via ELISA. Production of TNF and IGF-1 was decreased in both knockouts compared with WT regardless of the stimulus utilized, whereas IL-6 and VEGF levels appeared to be cooperatively regulated by both the activated TNF receptor and the initial stimulus. IL-6 was increased compared with WT in both knockouts following TNF stimulation but was significantly decreased with LPS. Compared with WT, hypoxia increased IL-6 in TNFR1KO but not TNFR2KO cells. TNF stimulation decreased VEGF in TNFR2KO cells, whereas TNFR1 ablation resulted in no change in VEGF compared with WT. TNFR1 ablation resulted in a decrease in VEGF following LPS stimulation compared with WT; no change was noted in TNFR2KO cells. With hypoxia, TNFR1KO cells expressed more VEGF compared with WT, whereas no difference was noted between WT and TNFR2KO cells. TNF receptor ablation modifies BMSC cytokine production. Identifying the proper stimulus and signaling cascades for the production of desired growth factors may be beneficial in maximizing the therapeutic potential of stem cells.     

Reversal of defective IL-6 production in lipopolysaccharide-tolerant mice by phorbol myristate acetate

The development of LPS tolerance has been suggested to be mediated by an inhibition of cytokine synthesis. Here we have studied serum IL-6 and TNF levels in mice after LPS administration. Repeated administration of LPS (35 micrograms daily for 4 days) to mice induced a refractoriness (tolerance) to subsequent administrations of LPS in terms of induction of circulating IL-6 and TNF. To investigate the mechanism by which LPS down-regulates its own induction of cytokine synthesis and the relationship between IL-6 and TNF production, we attempted to revert the inhibition of IL-6 and TNF production using agents like PMA or IFN-gamma, previously reported to activate macrophage production of cytokines. Pretreatment with PMA (4 micrograms, 10 min before LPS) partially restored IL-6 production in LPS-tolerant mice given 2 micrograms LPS. On the other hand, PMA did not restore TNF induction in LPS-tolerant mice, even when administered with high doses of LPS (up to 200 micrograms). A similar reversal of LPS resistance to IL-6, but not TNF, induction by PMA was observed in genetically LPS-resistant C3H/HeJ mice. IFN-gamma also restored, although to a lesser extent than PMA, IL-6 production. However, unlike PMA, IFN-gamma could also partially restore TNF production in LPS-tolerant mice, although only when LPS was administered at high doses. By contrast with PMA, IFN-gamma was clearly more active in restoring TNF synthesis than that of IL-6. Similar results were obtained in genetically LPS-unresponsive C3H/HeJ mice. These data suggest that different mechanisms are implicated in the inhibition of IL-6 and TNF synthesis in LPS-tolerant mice and that part of this inhibition can be overcome by PMA or IFN-gamma.