Enhancement of macrophage colony-stimulating factor-induced growth and differentiation of human monocytes by interleukin-10

Interleukin-10 (IL-10) has been reported to be a negative cytokine for monocytes/macrophages. In the present study, we showed that IL-10 is rather a positive cytokine and augments the growth and differentiation of human monocytes stimulated with macrophage colony-stimulating factor (M-CSF). Highly purified adherent human monocytes were cultured for 7 days with M-CSF in the presence or absence of IL-10. The number of recovered cells increased in the culture of monocytes with M-CSF + IL-10 compared to the culture with M-CSF alone. IL-10 alone was not enough to maintain the survival and differentiation of monocytes into macrophages. Morphological change cultured in M-CSF was also accelerated by addition of IL-10, and macrophages cultured in M-CSF + IL-10 were more elongated compared to macrophages cultured with M-CSF alone. Binding of 125I-M-CSF to monocytes incubated with M-CSF + IL-10 was about 1.7-fold higher than that to monocytes incubated with M-CSF alone. In accordance with the binding study, Northern blot analysis showed that the levels of the expression of c-fms, M-CSF receptor, mRNA in macrophages cultured in M-CSF + IL-10 were higher than that in macrophages cultured in M-CSF alone. Macrophages cultured in M-CSF + IL-10 expressed higher level of Fc gamma RI, II, III, and showed augmented Fc gamma receptor mediated phagocytosis. The former also produced higher level of H2O2 and O2-, when stimulated with zymosan, and of IL-6 when stimulated with lipopolysaccharide compared to the latter. These results taken together suggest that IL-10 augments the growth and differentiation of human monocytes cultured in M-CSF.     

Heparin-binding protein (CAP37) is internalized in monocytes and increases LPS-induced monocyte activation

Previous studies have shown that the neutrophil-derived heparin-binding protein (HBP), also known as CAP37 or azurocidin, potentiates the LPS-induced release of proinflammatory cytokines (TNF-alpha, IL-1, and IL-6) from isolated human monocytes. To date, the mechanisms by which HBP enhances LPS-induced monocyte activation have not been elucidated, and it is not known whether HBP also increases the LPS-induced production of other bioactive substances. We studied human monocytes activated by recombinant human HBP and LPS and their interaction with the LPS receptor CD14. We hypothesized that the stimulatory effect of HBP on the LPS-induced release of proinflammatory mediators from monocytes was mediated by specific binding of HBP to monocytes, which resulted in an up-regulation of CD14. Our results demonstrated that HBP alone (10 microg/ml) stimulated the production of TNF-alpha from isolated monocytes. In addition, HBP had an additive effect on LPS-induced production of TNF-alpha and PGE2, suggesting a generalized monocyte activation. We used flow cytometry to demonstrate that HBP had a high affinity to monocytes but not to the LPS receptor CD14, and experiments performed at 4 degrees C indicated an energy-dependent step in this process. Confocal microscopy showed that monocytes internalize HBP within 30 min. These data suggest that mechanisms other than increased CD14 expression are responsible for the enhanced release of TNF-alpha or PGE2 in response to HBP and LPS.     

Interleukin-10 controls interferon-gamma and tumor necrosis factor production during experimental endotoxemia

Interleukin-10 (IL-10) is a potent inhibitor of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production and has been shown to protect mice from endotoxin shock. As IFN-gamma is another important mediator of LPS toxicity, we studied the effects of IL-10 on LPS-induced IFN-gamma synthesis in vitro and in vivo. First, we found that the addition of recombinant human IL-10 (rhIL-10) (10 U/ml) to human whole blood markedly suppressed LPS-induced IFN-gamma release while neutralization of endogenously synthesized IL-10 resulted in increased IFN-gamma levels. The ability of rIL-10 to inhibit LPS-induced IFN-gamma synthesis was also observed in vivo in mice. Indeed, administration of 1000 U recombinant mouse IL-10 (rmIL-10) 30 min before and 3 h after challenge of BALB/c mice with 100 micrograms LPS resulted in a threefold decrease in peak IFN-gamma serum levels. We then examined the production and the role of IL-10 during murine endotoxemia. We found that LPS injection causes the rapid release of IL-10, peak IL-10 serum levels being observed 90 min after LPS challenge. Neutralization of endogenously produced IL-10 by administration of 2 mg JES5-2A5 anti-IL-10 monoclonal antibody (mAb) 2 h before LPS challenge resulted in a marked increase in both TNF and IFN-gamma serum levels while irrelevant isotype-matched mAb had no effect. The enhanced production of inflammatory cytokines in anti-IL-10 mAb-treated mice was associated with a 60% lethality after injection of 500 micrograms LPS, while all mice pretreated with control mAb survived. We conclude that the rapid release of IL-10 during endotoxemia is a natural antiinflammatory response controlling cytokine production and LPS toxicity.     

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.     

Human macrophages induced in vitro by macrophage colony-stimulating factor are deficient in IL-12 production

IL-12 is important for Th1 differentiation. Myeloid-derived antigen-presenting cells (APC) such as monocytes, macrophages (Mphi) and dendritic cells (DC) are believed to be major sources of IL-12 in vivo. We have compared IL-12 production of fresh monocytes with Mphi differentiated in vitro using macrophage colony-stimulating factor (M-CSF) or human plasma, and in vitro generated dendritic cells, since these differentiated cell types represent APC at sites of antigen challenge. Macrophages stimulated with lipopolysaccharide (LPS) or heat-killed Listeria monocytogenes in the presence or absence of IFN-gamma produced minimal IL-12 p70 by comparison with DC or monocytes, despite comparable production of TNF-alpha. M-CSF-induced Mphi produced low levels of IL-10 constitutively and high levels after stimulation with LPS, but neutralization of IL-10 did not augment Mphi IL-12 production. Exposure of Mphi to TNF-alpha, granulocyte-macrophage CSF or IFN-gamma did not substantially up-regulate IL-12. Therefore M-CSF induces a differentiated Mphi phenotype in which IL-12 production is down-regulated, perhaps irreversibly. This may be the default pathway for monocyte-Mphi development in the absence of inflammation.     

Differential effects on TNF alpha production by pharmacological agents with varying molecular sites of action

This study describes the activation conditions for tumor necrosis factor-alpha (TNF alpha) production in myelomonocytic U937 cells and human primary peripheral blood monocytes in response to lipopolysaccharide (LPS) and/or phorbol 12-myristate 13-acetate (PMA). PMA itself induced only low levels of TNF alpha production with delayed kinetics (e.g. 0.758 +/- 0.128 ng/ml from U937 cells after 48 h) while LPS induced greater levels of TNF alpha production in less time (e.g. 2.083 +/- 0.96 ng/ml from monocytes in 24 h). Pharmacological agents with various molecular sites of action were used to validate the two systems, with the protein serine-threonine kinase inhibitors staurosporine and Ro-31-8220, the protein tyrosine kinase inhibitor herbimycin A (HBA) and dexamethasone exhibiting the greatest potency (IC50S 5-350 nM). In contrast to the effect on TNF alpha production, PMA induced strong phosphorylation/activation of p42/p44mapk in monocytes by 10 min determined in a mobility shift assay, while LPS was a weaker inducer. Additionally, staurosporine (to LPS and PMA) and HBA (to LPS only) inhibited the activation of these mitogen-activated protein kinase (MAPK) isoforms at doses 10-100 fold higher than those required to inhibit maximal TNF alpha production. These data indicate the involvement of the p42/p44mapk signalling pathway in LPS-induced pro-inflammatory cytokine production but suggest that other signalling pathways are also implicated in this phenomenon.     

In vitro prevention and reversal of lipopolysaccharide desensitization by IFN-gamma, IL-12, and granulocyte-macrophage colony-stimulating factor

LPS tolerance is characterized by a diminished monocytic synthesis of TNF-alpha and, interestingly, IL-10 after LPS restimulation. We wondered whether granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-12, and IFN-gamma can prevent or reverse this down-regulation of TNF-alpha and IL-10 production. The LPS-induced TNF-alpha amounts in desensitized PBMC treated with GM-CSF, IFN-gamma, or IL-12 and in naive, non-cytokine-primed cultures were similar, while much more TNF-alpha was induced in cytokine-primed naive cells. The effect of IL-12 was dependent on the presence of nonmonocytic cells and could be completely blocked with an IFN-gamma antiserum. Treatment of LPS-desensitized pure monocytes with IFN-gamma or GM-CSF resulted in a very high TNF-alpha expression and no difference to cytokine-primed naive monocytes was evident any longer. While IFN-gamma and IL-12 decreased IL-10 expression in naive PBMC, it was increased by both and by GM-CSF in LPS-tolerant cultures. Again, only IL-12 was dependent on the presence of nonmonocytic cells. For prevention of LPS tolerance, similar results were obtained. Recently, we have shown that IL-10 and TGF-beta mediate LPS desensitization in vitro and can be used to establish LPS hyporesponsiveness in the absence of LPS. IFN-gamma and GM-CSF prevented and reversed down-regulation of TNF-alpha and IL-10 synthesis also in the model of IL-10/TGF-beta1-induced LPS hyporesponsiveness, while IL-12 was ineffective because of its obvious inability to induce IFN-gamma. In summary, after LPS desensitization/hyporesponsiveness, IFN-gamma and GM-CSF tended to normalize pro- and anti-inflammatory monocytic behavior. Our results suggest that during LPS desensitization/hyporesponsiveness, monocytes acquire a hitherto unknown functional state with an altered reaction to biologic response modifiers.     

Monocyte B7 and Sialyl Lewis X modulates the efficacy of IL-10 down-regulation of LPS-induced monocyte tissue factor in whole blood

Recent studies have investigated the use of anti-inflammatory cytokine, interleukin 10 (IL-10) to control the development of disseminated intravascular coagulation (DIC) in sepsis by down-regulation of monocyte tissue factor (MTF) induced by lipopolysaccharide (LPS) in the initial phase of the disease. In vitro and in vivo human studies have shown that a minimal (<1 h) delay in IL-10 treatment significantly reduces the cytokines ability to inhibit LPS-induced MTF expression and the end products of coagulation. In this whole blood in vitro study we investigated the role of lymphocyte and platelet interactions with monocytes to up-regulate MTF expression in the presence of IL-10 in the initial phase of exposure to LPS. Individual blockade of monocyte B7 or platelet P-selectin significantly (35%) reduced MTF expression (P<0.05). IL-10 showed a dose-dependent inhibition of LPS (0.1 microg/ml) induced MTF expression, with 56% inhibition at 1 ng/ml, maximizing at 5 ng/ml IL-10 (75%; P<0.05). Simultaneous exposure to LPS and IL-10 (1 ng/ml) or addition of IL-10 1 h after LPS, with individual B7 and P-selectin blockade significantly enhanced the inhibition of MTF expression by IL-10 (P<0.05). We conclude that the efficacy of IL-10 to control DIC could be enhanced by a simultaneous B7 and P-selectin blockade.     

Regulation of neutrophil-derived IL-8: the role of prostaglandin E2, dexamethasone, and IL-4

Historically, the neutrophil has been perceived as a terminally differentiated leukocyte with limited ability to produce de novo proteins. Furthermore, in the context of acute inflammation the activated neutrophil has been appreciated only for its ability to release various proteases, reactive oxygen, and arachidonic acid metabolites. Recently, the neutrophil has been shown to have the capacity to produce a number of cytokines that may be instrumental in orchestrating the progression of acute inflammation to a more chronic and specific immune response. These cytokines include IFN-alpha, M-CSF, G-CSF, TNF, IL-1, and IL-6. Our laboratory and others have shown that neutrophils produce IL-8 in response to LPS or a phagocytic challenge. Although these studies have shown the induction of IL-8 from polymorphonuclear neutrophils (PMN), relatively little is known regarding the regulation of PMN-derived IL-8. Because PMN and monocytes share the same stem cell, and monocyte-derived IL-8 is regulated by prostaglandin E2 (PGE2), glucocorticoids (dexamethasone; DEX) and the T-Lymphocyte-derived IL-4, we postulated that PMN-derived IL-8 production may be regulated in a similar manner. To test this hypothesis, PMN were isolated (> 99% pure) from peripheral blood and cultured in media with 5% FCS in the presence or absence of LPS (10 ng/ml; a concentration of LPS that induced the half-maximal production of PMN-derived IL-8) and in the presence or absence of DEX (10(-6) M to 10(-10) M), PGE2 (10(-6) M to 10(-10) M), or IL-4 (100 ng/ml to 100 pg/ml). PMN-derived IL-8 was measured using a specific sandwich ELISA. DEX and IL-4 in the presence of LPS were found to inhibit PMN-derived IL-8 in both a dose- and time-dependent fashion. DEX and IL-4 in concentrations of 10(-6) M and 10 ng/ml resulted in maximal inhibition of LPS-induced PMN-derived IL-8, respectively. Moreover, both DEX and IL-4 administration could be delayed 4 hr post-stimulation with LPS and result in significant suppression of PMN-derived IL-8. Interestingly, in contrast to the regulation of monocyte-derived IL-8 by PGE2, PGE2 treatment of PMN failed to inhibit the generation of LPS-induced IL-8. Northern blot analysis of steady-state IL-8 mRNA demonstrated that both DEX and IL-4 treatment of PMN resulted in a 40 and 52% reduction in LPS-stimulated PMN-derived IL-8 mRNA, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of nitric oxide synthase attenuates lipopolysaccharide-induced fever without reduction of circulating cytokines in guinea-pigs

It was recently demonstrated that the diffusible messenger molecule nitric oxide (NO) is involved in the febrile response of rats and rabbits to exogenous or endogenous pyrogens. In this study we have investigated the effects of systemic administration of the NO-synthase inhibitor N-nitro-l-arginine-methylester (l-NAME) on abdominal temperature and on lipopolysaccharide- (LPS-) induced fever in guinea-pigs. We further studied the effects of l-NAME on the LPS-induced circulating cytokine network by measurement of tumor necrosis factor alpha (TNF) and interleukin-6 (IL-6) in blood plasma during the time course of fever. At a dose of 10 mg/kg, intra-arterial injection of l-NAME per se had no influence on the abdominal temperature of guinea-pigs, while administration of 50 mg/kg l-NAME evoked a pronounced fall of body temperature which lasted about 12 h. When injected simultaneously with 10 microgram/kg LPS into the arterial circulation, the lower dose of l-NAME that did not decrease abdominal temperature per se caused a significant attenuation of LPS-induced fever due to suppression of the second phase of the biphasic febrile response. The LPS-induced cytokine network remained unimpaired by the treatment with l-NAME. Peak activity of TNF in plasma (measured 60 min after LPS injection) was 20,596+/-2368 pg/ml in control animals and 18,900+/-4778 pg/ml in guinea-pigs treated with l-NAME. In addition, circulating levels of IL-6 were not statistically different between both groups of animals 60 min or 180 min after administration of LPS along with l-NAME or vehicle. The results confirm that endogenous NO formation has a role in the generation of LPS-induced fever and demonstrate that the attenuation of fever by inhibition of NO-synthase is independent of the circulating LPS-induced cytokine network.     

Involvement of the IL-2 receptor gamma-chain (gammac) in the control by IL-4 of human monocyte and macrophage proinflammatory mediator production

IL-4 has potent anti-inflammatory properties on monocytes and suppresses both IL-1beta and TNF-alpha production. Well-characterized components of the IL-4 receptor on monocytes include the 140-kDa alpha-chain and the IL-2R gamma-chain, gammac, which normally dimerize 1:1 for signaling from the receptor. However, mRNA levels for gammac were very low in 7-day-cultured monocytes. As mRNA levels for gammac declined with culture, so too did the ability of IL-4 to down-regulate LPS-induced TNF-alpha production. In contrast, IL-4 consistently down-regulated IL-1beta production by cultured monocytes. Immunoprecipitation and Western blot analyses demonstrated that 7-day-cultured monocytes do not express the functionally active 64-kDa gammac protein. This was associated with decreased STAT6 activation by IL-4. Studies with Abs to gammac and an IL-4 mutant that is unable to bind to gammac showed that IL-4 can suppress IL-1beta but not TNF-alpha production by LPS-stimulated monocytes in the presence of little or no functioning gammac. IL-4 also suppressed IL-1beta but not TNF-alpha production by Mono Mac 6 cells, which express minimal levels of gammac. For gammac-expressing LPS/PMA-activated U937 cells, IL-4 decreased both TNF-alpha and IL-1beta production. These results suggest that functional gammac is not present on in vitro-derived macrophages, and that while some anti-inflammatory responses to IL-4 are lost with this down-regulation of functional gammac, others are retained. We conclude that different functional responses to IL-4 by human monocytes and macrophages are regulated by different IL-4 receptor configurations.     

Coordinate and noncoordinate colony stimulating factor formation by human monocytes

The regulation of macrophage colony-stimulating factor (M-CSF) formation by elutriation-purified human monocytes was studied in vitro and compared with that for granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF). The levels of the three CSFs were measured by immunoassay. Lipopolysaccharide (LPS, 100 ng/ml) was able to enhance CSF formation but the levels were significantly influenced by the presence of a cyclooxygenase product(s) in the cultures. In the presence of LPS, both M-CSF and GM-CSF were up-regulated by cyclooxygenase inhibition (indomethacin, 10(-5) M), while G-CSF was down-regulated. Exogenous prostaglandin E2 (PGE2, 10(-7) M) reversed the actions of indomethacin. In LPS-treated cells, in contrast to the different regulation by endogenous eicosanoid of M-CSF and GM-CSF formation and G-CSF formation, both interleukin-4 (IL-4, 250 pM) and the glucocorticoid dexamethasone (10(-7) M) lowered the amounts of all CSFs. When the actions of CSFs were examined for their effects on CSF formation, M-CSF could not stimulate either GM-CSF or G-CSF synthesis, in contrast to literature findings, while GM-CSF enhanced M-CSF formation but not that of G-CSF. These studies indicate that there can be both coordinate and noncoordinate control of CSF expression by human monocytes.     

IL-10 inhibits LPS-induced human monocyte tissue factor expression in whole blood

Interleukin 4 (IL-4), IL-10 and IL-13 are all known to modulate several proinflammatory functions in human monocytes. They have also previously been shown to down-regulate lipopolysaccharide (LPS)-induced tissue factor (TF) expression in isolated cultured monocytes. In this study we investigated the effect of these three cytokines on the induction of monocytic TF in a whole blood environment at three levels: mRNA quantitation, surface antigen expression and procoagulant activity. We showed that IL-10 attenuated LPS-induced monocyte TF expression and activity in whole blood in a concentration-dependent manner, both when added to the blood prior to LPS and, although to a lesser extent, when added up to 1 h subsequent to LPS challenge. Maximum inhibition occurred at 5 ng/ml of IL-10 when the cytokine was added before LPS. IL-4 and IL-13, however, did not exhibit any inhibitory effect in the whole blood environment, contrary to the reported findings in cell culture experiments. Our results confirm the potential of IL-10 as an anti-inflammatory, TF-preventing drug, whereas the effects of IL-4 and IL-13 on monocytes in whole blood seem more complex, and require further investigation.     

Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-alpha: role of the p38 and p42/44 mitogen-activated protein kinases

IL-10 is an anti-inflammatory cytokine with potent immunomodulatory effects, including inhibition of cytokine production. However, regulation of monocyte IL-10 production is poorly understood. In this report we have investigated the mechanisms of LPS-induced IL-10 production by human peripheral blood monocytes and demonstrate that IL-10 synthesis is uniquely dependent on the endogenous proinflammatory cytokines IL-1 and/or TNF-alpha. LPS signal transduction in monocytes has been shown to involve activation of the p38 and p42 mitogen-activated protein kinase (MAPK) cascades. The results in this paper indicate that inhibition of p38 MAPK potently inhibited the production of IL-10, IL-1beta, and TNF-alpha, whereas blockade of the p42/44 MAPK pathway, while partially inhibiting TNF-alpha and IL-1beta production, had no effect on monocyte secretion of IL-10. Furthermore, neither the inhibition of monocyte TNF-alpha induced by IL-10 nor the stimulation of soluble TNF receptor production was affected by inhibition of the p42/44 MAPK pathway, suggesting that this signaling event is not involved in either monocyte production of or anti-inflammatory responses to IL-10. These data raise the interesting possibility that proinflammatory TNF-alpha-mediated effects may be selectively blocked without modulating the induction or the response to IL-10, whereas the signaling events associated with the anti-inflammatory events induced by IL-10 remain to be elucidated.     

Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide

Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.     

Interleukin-10 inhibits activation of coagulation and fibrinolysis during human endotoxemia

Interleukin-10 (IL-10) has been found to inhibit lipopolysaccharide (LPS)-induced tissue factor expression by monocytes in vitro. To determine the effects of IL-10 on LPS-induced activation of the hemostatic mechanisms in vivo, we performed a placebo-controlled, cross-over study of human endotoxemia. Two groups of eight volunteers were challenged with LPS (4 ng/kg) on two occasions: once in conjunction with placebo, and once with recombinant human IL-10 (rhIL-10; 25 microg/kg). In group 1, placebo or rhIL-10 was given 2 minutes before LPS challenge, group 2 received placebo or rhIL-10 1 hour after LPS administration. Pretreatment with rhIL-10 reduced both LPS-induced activation of the fibrinolytic system (plasma concentrations of tissue type plasminogen activator, plasmin-alpha2-antiplasmin complexes, and D-dimer), and inhibition of fibrinolysis (plasma levels of plasminogen activator inhibitor 1), whereas posttreatment only inhibited the latter response. Both IL-10 pre- and posttreatment attenuated activation of the coagulation system (plasma levels of prothrombin fragment F1 + 2 and thrombin-antithrombin complexes). These results indicate that rhIL-10, besides its well-described inhibitory effects on cytokine release, potently modulates the fibrinolytic system and inhibits the coagulant responses during endotoxemia.     

Bacillus Calmette-Guérin potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor and interleukin-1, but not interleukin-6 in bladder cancer patients

During the past decade, particular attention has been focused on treatment of bladder cancer patients with the bacterial agent bacillus Calmette-Guérin (BCG). In these studies, bladder cancer patients were instilled with BCG (75 mg/50 ml) once per week for 6 weeks, 1-2 weeks following trans-urethral resection of the bladder. Cystoscopy was performed after 6 weeks and, unless tumor progression was present, monthly treatments were given for 1 year. Blood was drawn 2 h after the last instillation, and monocytes were isolated (5 x 10(6) cells/ml) and treated, or not, with lipopolysaccharide (LPS) (20 microgram/ml) for tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) release. The levels of monokines were determined by a monokine-specific enzyme-linked immunosorbent assay. Our results clearly show that, after 18 h incubation, macrophages from BCG-treated bladder cancer patients produced from 2.8- to 1.9-fold and from 2.0- to 1.3-fold greater amounts of TNF alpha and IL-1 alpha respectively, compared to macrophages from healthy controls, 5-fold higher than bladder cancer patients not treated with BCG. IL-6 was not affected. In another set of experiments macrophages (5 x 10(6) cells/ml) from healthy subjects were pretreated, or not, with BCG (100 micrograms/ml) overnight and treated, or not, with LPS 20 microgram/ml alone and in combination with interleukin-1 receptor antagonist (IL-1ra) 250 ng/ml. Macrophages treated with BCG had a strong stimulatory effect on IL-1 alpha release (9.45 ng/ml) while LPS was less effective (3.59 ng/ml). The combination of BCG plus LPS produced an additive effect on IL-1 alpha release (13.71 ng/ml) compared to the effect of the compound alone. The addition of IL-1ra (250 ng/ml) to BCG was not effective, while when IL-1ra was added to BCG plus LPS only a partial inhibition of IL-1 alpha release was found (9.83 ng/ml), compared to BCG plus LPS without IL-1ra (13.71 ng/ml). These effects seem to be related to the inhibition of IL-1 alpha stimulated with LPS, but not BCG. The priming effect of BCG exerted on LPS-stimulated monocyte production of TNF alpha and IL-1 alpha from bladder cancer patients led us to study the possible modulation of fibrinogen and C-reactive protein in the serum of BCG-treated cancer patients. The plasma levels of fibrinogen and C-reactive protein were higher (approximately twice) in BCG-treated patients compared to values obtained in untreated patients or healthy controls. We conclude that the beneficial immunotherapeutic effects of BCG in bladder cancer patients are related to its capacity to prime macrophages to enhance the release of TNF alpha and IL-1 alpha, but not IL-6 in response to physiological secondary stimuli, or through the direct stimulation of BCG on IL-1 alpha or TNF alpha, which are directly involved in the killing of cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)