Bikunin inhibits lipopolysaccharide-induced tumor necrosis factor alpha induction in macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-alpha) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-alpha expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-alpha protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 microM). (iii) Inhibition by bikunin of TNF-alpha induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-alpha target molecules interleukin-1beta (IL-1beta) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-alpha release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-alpha production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.     

Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A

Monophosphoryl lipid A (MPL) is a nontoxic derivative of lipopolysaccharide (LPS) that exhibits adjuvant properties similar to those of the parent LPS molecule. However, the mechanism by which MPL initiates its immunostimulatory properties remains unclear. Due to the involvement of Toll-like receptors in recognizing and transducing intracellular signals in response to LPS, the aim of the present study was to determine the ability of MPL to utilize the Toll-like receptor 2 (TLR2) and TLR4. We provide evidence that MPL differentially utilizes TLR2 and TLR4 for the induction of tumor necrosis factor alpha, interleukin 10 (IL-10), and IL-12 by purified human monocytes as well as by human peripheral blood mononuclear cells. Assessment of NF-kappa B activity demonstrated that MPL utilized TLR2 and especially TLR4 for the activation of NF-kappa B p65 by human monocytes. In addition, stimulation of human monocytes by MPL led to an up-regulation of the costimulatory molecules CD80 and CD86, an effect that could be reduced by pretreatment of cells with a monoclonal antibody to TLR2 or TLR4. Analysis of MPL-induced activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases revealed that MPL utilized both TLR2 and TLR4 for the phosphorylation of ERK1/2, while TLR4 was the predominant receptor involved in the ability of MPL to phosphorylate p38. Moreover, using selective inhibitors for MAP kinase kinase (PD98059) and p38 (SB203580), we show that ERK1/2 exhibited differential effects on production of TNF-alpha and IL-12 p40 by human monocytes, whereas MPL-induced activation of p38 appeared to be predominantly involved in production of IL-10 and IL-12 p40 by MPL-stimulated monocytes. Taken together, these findings aid in understanding the cellular mechanisms by which MPL induces host cell activation and subsequent adjuvant properties.     

Bikunin Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Induction in Macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-α) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-α expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-α protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 μM). (iii) Inhibition by bikunin of TNF-α induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-α target molecules interleukin-1β (IL-1β) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-α release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-α production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.     

Differential role of mitogen-activated protein kinases in CD40-mediated IL-12 production by immature and mature dendritic cells

Using a murine spleen-derived dendritic cell (DC) line (BC1) CD40-mediated interleukin (IL)-12 production was analyzed and compared between immature and mature DC. BC1 cells, immature DC (iDC), were maturated by treatment with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha. IL-12 production of LPS-treated DC (LPS/DC) was markedly enhanced by treatment with an anti-CD40 monoclonal antibody (mAb). Although the anti-CD40 mAb also enhanced IL-12 productions of iDC and TNF-alpha-treated DC (TNF/DC), these production levels were considerably low compared with that of LPS/DC. CD40-mediated IL-12-productions by iDC and TNF/DC were significantly enhanced by treatment with PD98059, a specific inhibitor of extracellular signal-related kinase (ERK) pathway. In contrast, PD98059 showed no significant effects on CD40-mediated IL-12-production by LPS/DC. These results demonstrated that ERK pathway was involved in negative regulation of the IL-12 productions by iDC and TNF/DC but not by LPS/DC. On the other hand, SB203580, a specific inhibitor of p38 mitogen activated protein kinase (MAPK) pathway, completely inhibited CD40-mediated IL-12-production by iDC, while not affecting those of TNF/DC and LPS/DC. Thus, p38 MAPK pathway appeared to positively regulate the IL-12 production in iDC but not in mature DC. It seems that roles of ERK and p38 MAPK for IL-12 production are developmentally changed in murine DC.     

Enhanced macrophage responsiveness to lipopolysaccharide and CD40 stimulation in a murine model of inflammatory bowel disease: IL-10-deficient mice

OBJECTIVE: To elucidate the role of macrophages in the pathogenesis of inflammatory bowel disease, proinflammatory characteristics of macrophages were estimated in a murine model of spontaneous intestinal inflammation. MATERIALS AND METHODS: Peritoneal macrophages from IL-10deficient mice were stimulated with lipopolysaccharide (LPS) or an anti-CD40 monoclonal antibody (mAb). Cytokine release was assessed by enzyme-linked immunosorbent assay. CD40 expression was examined by two-color flow cytometric analysis. Induction of suppressor of cytokine signaling 3 (SOCS3) mRNA was evaluated by real-time quantitative RT-PCR. RESULTS: In the presence of LPS or anti-CD40 mAb, TNF-alpha and IL-12p70 release from macrophages of mutant mice was significantly higher than that from macrophages of wild-type mice. This may be due to the difference in IL-10 production by macrophages, since activated macrophages of wild-type mice produced IL-10 in amounts sufficient to suppress an increased release of cytokines from activated macrophages of mutant mice. LPS and CD40 stimulation induced significantly high level of SOCS3 expression in macrophages of mutant mice in comparison to those of wild-type mice. CONCLUSIONS: Macrophages from a murine model of inflammatory bowel disease demonstrated enhanced responsiveness to immunological and bacterial stimuli. This suggests significant roles of macrophages in the pathogenesis of inflammatory bowel disease.     

The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

Induction of endotoxin tolerance in vivo inhibits activation of IRAK4 and increases negative regulators IRAK-M, SHIP-1, and A20

TLRs mediate host defense against microbial pathogens by eliciting production of inflammatory mediators and activating expression of MHC, adhesion, and costimulatory molecules. Endotoxin tolerance limits excessive TLR-driven inflammation during sepsis and reprograms macrophage responses to LPS, decreasing expression of proinflammatory cytokines without inhibiting anti-inflammatory and antimicrobial mediators. Molecular mechanisms of reprogramming of TLR4 signaling upon in vivo induction of endotoxin tolerance are incompletely understood. We used an in vivo model of endotoxin tolerance, whereby C57BL/6 mice were i.p.-inoculated with LPS or PBS, followed by in vitro challenge of peritoneal or splenic macrophages with LPS to examine activation of IRAK4 and expression of negative regulatory molecules. Administration of LPS in vivo-induced endotoxin tolerance in peritoneal and splenic macrophages, as evidenced by decreased degradation of IκBα, suppressed phosphorylation of p38 and reduced expression of TNF-α, IL-6, and KC mRNA upon in vitro LPS challenge. Macrophages from control and endotoxin-tolerant mice exhibited comparable TLR4 mRNA levels and similar expression of IL-1RA and IL-10 genes. Endotoxin tolerization in vivo blocked TLR4-driven IRAK4 phosphorylation and activation in macrophages, while increasing expression of IRAK-M, SHIP-1, A20 mRNA, and A20 protein. Thus, induction of endotoxin tolerance in vivo inhibits expression of proinflammatory mediators via impaired activation of IRAK4, p38, and NF-κB and increases expression of negative regulators of TLR4 pathways.     

Mechanism of crosstalk inhibition of IL-6 signaling in response to LPS and TNFalpha

Negative regulation of cytokine signaling is critical for the generation of the appropriate cellular outcome in response to signals, and can be modulated by other concomitant extracellular stimuli ("crosstalk"). Using both genetic and pharmacological manipulations we have investigated the mechanisms by which the pro-inflammatory stimuli, lipopolysaccharide (LPS) and Tumor necrosis factor alpha (TNFalpha), negatively regulate interleukin-6 (IL-6) signaling in primary mouse macrophages. Analysis of suppressor of cytokine signalling 3 (SOCS3)-deficient macrophages reveal that SOCS3 is necessary but surprisingly, not sufficient for the complete crosstalk inhibition of IL-6 signaling induced by LPS and TNFalpha. Analysis of macrophages from gp130 (Y757F) mutant mice suggest that SH2 domain-containing tyrosine phosphatase (SHP2) activity does not explain the residual inhibitory effect of these pro-inflammatory stimuli. In addition, p38 mitogen-activated protein kinase (p38) activation also negatively regulates IL-6 signaling independent of its parallel and necessary action to induce SOCS3 expression. Finally, we have identified an additional, novel mechanism of crosstalk inhibition: a reduction in total cellular levels of gp130 following stimulation with LPS and TNFalpha.     

Intracellular signaling mechanisms regulating the activation of human?eosinophils by the novel Th2 cytokine IL-33: implications for allergic?inflammation

The novel interleukin (IL)-1 family cytokine IL-33 has been shown to activate T helper 2 (Th2) lymphocytes, mast cells and basophils to produce an array of proinflammatory cytokines, as well as to mediate blood eosinophilia, IgE secretion and hypertrophy of airway epithelium in mice. In the present study, we characterized the activation of human eosinophils by IL-33, and investigated the underlying intracellular signaling mechanisms. IL-33 markedly enhanced eosinophil survival and upregulated cell surface expression of the adhesion molecule intercellular adhesion molecule (ICAM)-1 on eosinophils, but it suppressed that of ICAM-3 and L-selectin. In addition, IL-33 mediates significant release of the proinflammatory cytokine IL-6 and the chemokines CXCL8 and CCL2. We found that IL-33-mediated enhancement of survival, induction of adhesion molecules, and release of cytokines and chemokines were differentially regulated by activation of the nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we compared the above IL-33 activities with two structurally and functionally related cytokines, IL-1β and IL-18. IL-1β, but not IL-18, markedly upregulated cell surface expression of ICAM-1. IL-1β and IL-18 also significantly enhanced eosinophil survival, and induced the release of IL-6 and chemokines CXCL8 and CCL2 via the activation of the NF-κB, p38 MAPK and ERK pathways. Synergistic effects on the release of IL-6 were also observed in combined treatment with IL-1β, IL-18 and IL-33. Taken together, our findings provide insight into IL-33-mediated activation of eosinophils via differential intracellular signaling cascades in the immunopathogenesis of allergic inflammation.     

Brucella abortus as a potential vaccine candidate: induction of interleukin-12 secretion and enhanced B7.1 and B7.2 and intercellular adhesion molecule 1 surface expression in elutriated human monocytes stimulated by heat-inactivated B. abortus.

Development of a vaccine which is capable of generating a strong cellular immune response associated with gamma interferon (IFN-gamma) production and cytotoxic T-cell development requires that the immunogen be capable of inducing the secretion of interleukin-12 (IL-12), which is a pivotal factor for the differentiation of Th1 or Tc1 cells. We have previously shown that the heat-inactivated gram-negative bacterium Brucella abortus can induce IFN-gamma secretion by T cells. In the present study, we demonstrate that B. abortus and lipopolysaccharide (LPS) from B. abortus can induce IL-12 p40 mRNA expression and protein secretion by human elutriated monocytes (99% pure). p40 mRNA was detected within 4 h, and p40 protein could be measured at 24 h. This induction was abrogated by anti-CD14 monoclonal antibody, suggesting that monocytes recognize B. abortus via their receptor for LPS. The biological activity of IL-12 secreted by B. abortus-stimulated monocytes was demonstrated by its ability to upregulate IFN-gamma mRNA expression in T cells separated from monocytes and B. abortus by a transwell membrane. The B. abortus-induced IL-12 also enhanced NK cytolytic activity against K562 target cells. B. abortus was shown to rapidly increase the expression of the costimulatory molecules B7.1 and B7.2 and intercellular adhesion molecule 1 on human monocytes. Together, these data indicate that B. abortus can directly activate human monocytes and provide the cytokine milieu which would direct the immune response towards Th1-Tc1 differentiation.     

Activation of eosinophils by IL-12 family cytokine IL-27: Implications of the pleiotropic roles of IL-27 in allergic responses

Interleukin (IL)-27 is a novel IL-12 family cytokine and its immunomodulatory effects on T-lymphocytes, B-lymphocytes and mast cells were extensively studied. IL-27 could suppress Th2-mediated allergic diseases in mouse models. However, the role of IL-27 on eosinophils, the principal effector cells in allergic diseases, remains unexplored. Our present study revealed that eosinophils constitutively express functional IL-27 receptor heterodimer, gp130 and WSX-1. IL-27 could prolong eosinophil survival by reducing apoptosis, modulate the expression of adhesion molecules to facilitate eosinophil adhesion and accumulation, and induce the release of proinflammatory cytokines IL-6, tumor necrosis factor-α, IL-1β and chemokines CCL2, CXCL8 and CXCL1. The stimulation effects of IL-27 on eosinophils could not be abrogated by Th2 cytokine IL-25, hematopoietic cytokine granulocyte macrophage colony-stimulating factor, and toll-like receptor 4 ligand lipopolysaccharide (LPS). These findings are different from the effects of IL-27 and LPS on monocytes. Intracellular signaling mechanistic studies showed that IL-27-mediated eosinophil activation was differentially regulated by the activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, as well as nuclear factor-κB. Based on the above results, IL-27 could play crucial roles in allergic diseases by the activation of eosinophils via differential intracellular signaling cascades. According to the present findings of its activating effects on human eosinophils, IL-27 may play pleiotropic roles in human allergic responses.     

Soluble peptidoglycan-induced monokine production can be blocked by anti-CD14 monoclonal antibodies and by lipid A partial structures.

We have investigated the interaction of soluble peptidoglycan (sPG), in comparison with lipopolysaccharide (LPS), with human mononuclear cells (MNC) by determining the capacity of sPG to induce interleukin-6 (IL-6) and IL-1 release. In addition, we investigated the modulation of their interaction by anti-CD14 monoclonal antibody and by partial structures of LPS. We found that sPG, like LPS, was able to induce IL-6 and IL-1 production by MNC. However, dose-response experiments revealed that at least 3,000 ng of sPG per ml was necessary for induction, whereas the optimal LPS concentration was 1 ng/ml. Anti-CD14 monoclonal antibody reduced sPG- and LPS-induced IL-6 and IL-1 production. Moreover, partial structures of LPS were able to reduce monokine production induced by sPG and LPS. We conclude that sPG constitutes, like LPS, an inflammatory cytokine inducer and that CD14 is involved in the activation of human monocytes not only by LPS but also by sPG.     

Intracellular signaling mechanisms regulating toll-like receptor-mediated activation of eosinophils

Activation of eosinophils by microbe-derived molecules via Toll-like receptors (TLR) potentially provides the link between microbe-induced innate immune responses and the exacerbation of allergic inflammation. We investigated the expression of TLRs and the effect of their ligands on human eosinophils. Expression of TLR1-9 was detected by Western blot and flow cytometry. Adhesion molecules, cytokines, superoxides, and eosinophlilic cationic protein (ECP) were assessed by flow cytometry, enzyme-linked immunosorbent assay, chemiluminescent method, and fluorescence immunoassay, respectively. Human eosinophils differentially expressed TLR1, -2, -4, -5, -6, -7, and -9. Peptidoglycan (PGN) (TLR2 ligand), flagellin (TLR5 ligand), and Imiquimod R837 (TLR7 ligand) could significantly upregulate cell surface expression of intercellular adhesion molecule (ICAM)-1 and CD18, and induce the release of IL-1beta, IL-6, IL-8, growth-related oncogene (GRO)-alpha, and superoxides of eosinophils. Only PGN could induce the degranulation for ECP release. However, ds poly I-C (TLR3 ligand), LPS (TLR4 ligand), ssRNA (TLR8 ligand), and CpG-DNA (TLR9 ligand) were much less effective or inactive. PGN, flagellin, and R837 could activate both nuclear factor (NF)-kappaB and extracellular signal-regulated protein kinase (ERK). PGN could activate phosphatidylinositol 3-kinase (PI3K)-Akt, and R837 both PI3K-Akt and p38 mitogen-activated protein kinase (MAPK). The induction of the release of IL-1beta, IL-6, IL-8, GRO-alpha, superoxides, and ECP by PGN, flagellin, and R837 was found to be differentially regulated by NF-kappaB, ERK, PI3K-Akt, and p38 MAPK. The above results therefore support that microbial infection may lead to the exacerbation of allergic inflammation.     

Mechanism of crosstalk inhibition of IL-6 signaling in response to LPS and TNFα

Negative regulation of cytokine signaling is critical for the generation of the appropriate cellular outcome in response to signals, and can be modulated by other concomitant extracellular stimuli (“crosstalk”). Using both genetic and pharmacological manipulations we have investigated the mechanisms by which the pro-inflammatory stimuli, lipopolysaccharide (LPS) and Tumor necrosis factor α (TNFα), negatively regulate interleukin-6 (IL-6) signaling in primary mouse macrophages. Analysis of suppressor of cytokine signalling 3 (SOCS3)-deficient macrophages reveal that SOCS3 is necessary but surprisingly, not sufficient for the complete crosstalk inhibition of IL-6 signaling induced by LPS and TNFα. Analysis of macrophages from gp130 (Y757F) mutant mice suggest that SH2 domain-containing tyrosine phosphatase (SHP2) activity does not explain the residual inhibitory effect of these pro-inflammatory stimuli. In addition, p38 mitogen-activated protein kinase (p38) activation also negatively regulates IL-6 signaling independent of its parallel and necessary action to induce SOCS3 expression. Finally, we have identified an additional, novel mechanism of crosstalk inhibition: a reduction in total cellular levels of gp130 following stimulation with LPS and TNFα.     

Regulation of TREM expression in hepatic macrophages and endothelial cells during acute endotoxemia

Triggering receptor expressed on myeloid cells (TREM) regulates inflammatory responses to lipopolysaccharide (LPS). In these studies, we analyzed the expression of TREM in hepatic macrophages and endothelial cells which play a central role in LPS clearance. LPS administration to C3H/HeOuJ mice resulted in a rapid induction of TREM-1 and TREM-3, but a decrease in TREM-2 in liver macrophages and endothelial cells. The observation that TREM family members are detectable in endothelial cells is novel and demonstrates that their expression is not limited to myeloid cells. LPS-induced alterations in TREM expression were not evident in cells from C3H/HeJ TLR-4 mutant mice, indicating that the response is dependent on TLR-4. IL-1beta and TNFalpha upregulated TREM-1 and TREM-3 expression and suppressed TREM-2 expression in macrophages and endothelial cells. This activity involved PI3-kinase and p38 MAP kinase signaling. Interestingly, no significant differences were noted in TREM expression between wild-type and TNFR1-/- mice treated with LPS. Treatment of macrophages and endothelial cells with LPS upregulated expression of nitric oxide synthase-2 (NOS-2). This was blocked by TREM-1 Fc/fusion protein, indicating that TREM-1 mediates LPS-induced NOS-2 expression. These results suggest that TREM proteins are important in the inflammatory response of hepatic macrophages and endothelial cells to acute endotoxemia.     

Chlorpromazine specifically inhibits peripheral and brain TNF production, and up-regulates IL-10 production, in mice.

We have previously shown that chlorpromazine (CPZ) inhibits tumour necrosis factor (TNF) production and protects against endotoxic shock in mice. In this paper we investigated the effect of pretreatment with CPZ, 4 mg/kg i.p. 30 min before, compared with dexamethasone (DEX; 3 mg/kg) on the induction of other endotoxin (lipopolysaccharide; LPS)-induced cytokines in the serum of mice, i.e. interleukin-1 alpha (IL-1 alpha), IL-6 and IL-10, and TNF. We also studied the effect of CPZ on serum and spleen-associated TNF. Both DEX and CPZ inhibited TNF production, whereas induction of IL-1 and IL-6 was inhibited by DEX but not by CPZ. DEX did not affect IL-10, while CPZ potentiated its induction. CPZ also inhibited spleen-associated TNF induction in LPS-treated mice, suggesting an effect on the synthesis of TNF. CPZ inhibited TNF induction by Gram-positive bacteria (heat-killed Staphylococcus epidermidis) and by anti-CD3 monoclonal antibodies. Intraperitoneal administration of CPZ also inhibited the induction of brain-associated TNF induced by intra-cerebroventricular injection of LPS. Therefore, CPZ is a more specific inhibitor of TNF production than DEX; in particular, CPZ increased the induction of IL-10, which is a 'protective' cytokine known to inhibit LPS toxicity and TNF production. CPZ inhibited TNF production in vivo, irrespective of the TNF stimulus used to induce TNF. Finally, CPZ did not induce the 'rebound' effect of DEX that, when given 24 hr before LPS, potentiates TNF production, but it did inhibit TNF production after 24 hr.