Secretion of IL-1: role of protein kinase C.

In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC.     

Plasma membrane-associated tumor necrosis factor. A non-integral membrane protein possibly bound to its own receptor

Purified plasma membranes from LPS-activated human blood monocytes produced significant lysis and growth inhibition of the TNF-sensitive L929 murine fibroblast cell line. Unactivated human monocyte plasma membranes did not display either activity. Anti-TNF serum specifically inhibited the anti-tumor activity of activated monocyte membranes whereas anti-IL-1 serum or non-specific rabbit serum decreased neither the lysis nor growth inhibition of L929 cells. Membrane-associated TNF did not behave as an integral protein as it could be eluted from the plasma membranes by either high salt or low pH treatment. Plasma membranes cleared of membrane-associated TNF by high salt treatment were able to bind TNF, and this binding was specifically inhibited by preincubation of rTNF with specific anti-TNF serum. Western blot analysis of plasma membranes showed a membrane-associated TNF with a m. w. of approximately 17 kDa present only in the activated monocytes. When the plasma membranes were preincubated with the cross-linker agent dissuccinimidyl suberate, Western blot analysis revealed the presence of a TNF-binding protein with a Mr of approximately 102 kDa. These studies indicate that unlike IL-1, membrane-associated TNF is not an integral membrane protein and that TNF may be associated with the monocyte membrane by occupying the TNF R.     

Lipopolysaccharide and interleukin 1 inhibit interferon-gamma-induced Fc receptor expression on human monocytes

The objectives of these studies were to study the effects of bacterial lipopolysaccharide (LPS) on interferon-gamma (IFN-gamma)-induced Fc receptor expression on human monocytes and to examine whether these effects were mediated through stimulation of interleukin 1 (IL-1) production. Fc receptor expression was determined by binding of monomeric monoclonal murine immunoglobulin (Ig)G2a and cytofluorographic analysis. IL-1 activity in monocyte supernatants and lysates was assayed by augmentation of mitogen-induced murine thymocyte proliferation. IFN-gamma induced the expression of Fc receptors on human monocytes that were specific for murine IgG2a. This induction was inhibited by the addition of LPS in amounts as low as 2 to 8 pg/ml. LPS inhibition of IFN-gamma-induced Fc receptor expression was paralleled by the appearance of IL-1 in monocyte lysates and supernatants. The addition of purified human or recombinant IL-1 beta at the initiation of culture similarly inhibited the expression of IFN-gamma-induced Fc receptors on the monocytes. LPS also inhibited Fc receptor expression on the human myelomonocytic cell line THP-1 after induction with IFN-gamma or phorbol myristate acetate alone or with both agents together. This inhibition also was paralleled by the production of IL-1 but the addition of exogenous IL-1 to the THP-1 cells had no effect on IFN-gamma-induced Fc receptor expression. Tumor necrosis factor (TNF) inhibited IFN-gamma-induced Fc receptor expression on human monocytes but was much less potent than comparable amounts of IL-1. TNF also did not inhibit Fc receptor expression on THP-1 cells. In fact, IL-1 or TNF led to an enhancement in IFN-gamma-induced Fc receptor expression on THP-1 cells. These results indicate that LPS can inhibit IFN-gamma-induced Fc receptor expression on human monocytes and that IL-1 and TNF may mediate these effects of LPS. Thus, an autocrine or paracrine role is suggested for these cytokines. The possibility exists that intracellular IL-1 resulting from LPS stimulation may be at least in part responsible for inhibition of Fc receptor expression.     

Priming of neutrophils by lipopolysaccharide for enhanced release of superoxide. Requirement for plasma but not for tumor necrosis factor-alpha

When human neutrophils are incubated with LPS, they become primed for enhanced release of O2- in response to stimulation by FMLP. We investigated two aspects of LPS priming: 1) whether priming depends on secretion of TNF-alpha by monocytes present in neutrophil preparations, and 2) whether plasma is required for priming. Using plasma-Percoll gradients, we isolated neutrophils that contained only 0.1% monocytes. At 37 degrees C, these neutrophils were significantly primed by LPS (100 ng/ml) within 30 min. In contrast, LPS-treated monocytes required 60 min to secrete significant neutrophil-priming activity, the major component of which was TNF-alpha. Further, antibody against TNF-alpha failed to inhibit priming of neutrophils by LPS at 15, 30, and 45 min, and inhibited only 15% at 60 min. The results suggested that TNF-alpha or other factors from monocytes were not essential for priming of neutrophils by LPS. Neutrophils that had been washed free of plasma by centrifugation through 50% Percoll responded only weakly to LPS with respect to priming for enhanced O2- release and increased expression of alkaline phosphatase activity on the cell surface. Priming of washed neutrophils could be restored by adding back plasma (0.1 to 1.0%). This effect of plasma was not blocked by heating the plasma to 56 degrees C but was blocked at 100 degrees C. LPS priming could be blocked by polymyxin B, even in the presence of plasma. Thus, priming required both LPS and plasma. Neutrophils incubated with LPS in the absence of plasma were not primed by subsequent addition of plasma, but were primed by addition of plasma and LPS. Culture supernatants from neutrophils incubated with 20 ng/ml LPS in the absence of plasma failed to prime fresh neutrophils, but supernatants from neutrophils incubated with LPS in the presence of 1% plasma were able to prime fresh neutrophils. These results implied that neutrophils inactivated LPS and that plasma protected LPS from inactivation. Nevertheless, such inactivated LPS retained the ability to gel Limulus lysate at 10 pg/ml, and the ability to prime monocytes at 100 pg/ml. Thus, plasma prevented a neutrophil-specific inactivation of LPS.     

Neurosteroid levels are increased in vivo after LPS treatment and negatively regulate LPS-induced TNF production

Neuroactive steroids such as dehydroepiandrosterone sulfate and pregnenolone inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production. Corticosteroids not only inhibit TNF production but their levels are increased in vivo after endotoxin injection, thus representing a feedback system that limits TNF production. We wondered whether the same could be true for neuroactive steroids. Thus, the possibility that neuroactive steroids might be increased concomitantly to TNF induction in vivo in mice treated with LPS was investigated. Increased plasma and hippocampal levels of allopregnanolone (but not of dehydroepiandrosterone or pregnenolone) were found 90 min after LPS injection. Allopregnanolone and progesterone (IC50 10- 7 and 10- 9 M, respectively) also inhibited TNF production by mouse peritoneal macrophages in vitro at concentrations in the range of those detected in vivo. These findings suggest that neuroactive steroids may act as endogenous inhibitors of cerebral and systemic TNF production.     

Human toll-like receptor 2 mediates monocyte activation by Listeria monocytogenes, but not by group B streptococci or lipopolysaccharide

Human Toll like receptor (TLR) 2 has been implicated as a signaling receptor for LPS from Gram-negative bacteria and cell wall components from Gram-positive organisms. In this study, we investigated whether TLR2 can signal cell activation by the heat-killed group B streptococci type III (GBS) and Listeria monocytogenes (HKLM). HKLM, but not GBS, showed a time- and dose-dependent activation of Chinese hamster ovary cells transfected with human TLR2, as measured by translocation of NF-kappaB and induction of IL-6 production. A mAb recognizing a TLR2-associated epitope (TL2.1) was generated that inhibited IL-6 production from Chinese hamster ovary-TLR2 cells stimulated with HKLM or LPS. The TL2.1 mAb reduced HKLM-induced TNF production from human monocytes by 60%, whereas a CD14 mAb (3C10) reduced the TNF production by 30%. However, coadministrating TL2.1 and 3C10 inhibited the TNF response by 80%. In contrast to this, anti-CD14 blocked LPS-induced TNF production from monocytes, whereas anti-TLR2 showed no inhibition. Neither TL2.1 nor 3C10 affected GBS-induced TNF production. These results show that TLR2 can function as a signaling receptor for HKLM, possibly together with CD14, but that TLR2 is unlikely to be involved in cell activation by GBS. Furthermore, although LPS can activate transfected cell lines through TLR2, this receptor does not seem to be the main transducer of LPS activation of human monocytes. Thus, our data demonstrate the ability of TLR2 to distinguish between different pathogens.     

Tumor necrosis factor release from lipopolysaccharide-stimulated human monocytes: Lipopolysaccharide tolerance in vitro

Human peripheral blood monocytes secrete tumor necrosis factor (TNF) in response to stimulation with bacterial lipopolysaccharide (LPS). We have shown that isolated human monocytes pretreated with LPS for 24 h secrete lower levels of TNF on a second stimulation with LPS than monocytes that have been stimulated with a single dose of LPS either immediately after isolation or 24 h after isolation. The levels of TNF released by monocytes after the second stimulation with LPS are proportional to the LPS concentration over a range from 1 ng/mL to 10 micrograms/mL. Increasing concentrations of LPS used during the first 24-h stimulation induce greater suppression of TNF release after a second stimulation with LPS. After an initial stimulus of 10 micrograms/mL LPS, a second stimulation of monocytes even with 10 micrograms/mL LPS will result in TNF secretion similar to that of unstimulated cells. This in vitro tolerance apparently can be overcome by stimulating previously activated cells with phorbol myristate acetate. We have also shown that neither prostaglandin E2 nor dexamethasone added during the initial stimulation with LPS had an effect on the subsequent reduction in TNF release on a second stimulation of monocytes with LPS.     

Interleukin 1 release by human monocytes treated with liposome-encapsulated lipopolysaccharide

Liposome therapy offers a unique method of delivering antifungals, antiprotozoans, and macrophage-activating factors directly to reticuloendothelial cells. Although liposomes are entering clinical trials, their effect on Il 1 synthesis and release has yet to be determined. The beneficial, as well as possible pathological, effects of liposome therapy may be due to IL 1 released by reticuloendothelial cells. This study examined the effects of multilamellar phospholipid vesicles on the synthesis and release of IL 1 from human peripheral blood monocytes. Liposomes consisting of phosphatidylcholine and phosphatidylserine in molar ratio of 7:3 did not stimulate IL 1 release, and did not diminish the level of IL 1 release when monocytes were subsequently stimulated with LPS or Staphylococcus aureus. Surprisingly, LPS encapsulated in liposomes failed to stimulate IL 1 release from monocytes. This defect was limited to the release of IL 1, because monocytes stimulated with liposomes containing LPS had control levels of intracellular (cytosolic) and membrane IL 1. By contrast, LPS associated with lyophilized liposomes induced the secretion of IL 1 and behaved similarly to free LPS. These findings indicate that LPS activation of monocytes may involve not only the synthesis of IL 1 but activation of the secretory mechanism for IL 1, and that these two events could be independent, that the density of LPS molecules at the surface of the liposome may influence the degree of monocyte activation as measured by intracellular IL 1 synthesis, membrane accumulation, and secretion into the medium, and that intracellular lipids may not interfere with the secretory mechanism, because liposomes made with phospholipids differing in acyl chain length or the headgroup behaved identically.     

Involvement of tumor necrosis factor in cytotoxicity mediated by human monocytes

Human monocytes cultured in a specially prepared medium free of lipopolysaccharide (LPS) constitutively produced a small, though significant, amount of tumor necrosis factor (TNF). Upon addition of LPS, the amount produced remained constant until the LPS concentration reached 1-10 ng/ml, whereupon the production of TNF dramatically increased, eventually becoming 100-fold greater than when the LPS concentration was below 1 ng/ml. Priming the monocytes with recombinant interferon-gamma (rIFN-gamma) before LPS exposure resulted in a 2- to 10-fold increase in TNF production, the highest relative increase being obtained at lower LPS concentrations and in the absence of LPS. Monocyte-produced TNF appears to be the effector molecule in monocyte-mediated killing of some target cell types, since antiserum against recombinant TNF inhibited killing of both actinomycin D-treated and untreated WEHI 164 cells by human monocytes. However, it also appears that TNF may not in all cases be an effector molecule in monocyte-mediated killing, since cytolysis of K562 cells mediated by IFN-gamma/LPS-activated monocytes was not inhibited by antiserum against recombinant TNF. Antiserum which was raised against a monocyte-derived cytotoxic factor and which neutralized recombinant TNF did, however, inhibit monocyte-mediated cytolysis of K562 cells, suggesting that an extracellular factor, perhaps related to TNF, was also involved in monocyte-mediated killing of K562 cells. A TNF-like activity was associated with the monocyte surface membrane, since paraformaldehyde-fixed monocytes expressed cytotoxic activity which was neutralized by antiserum against recombinant TNF. Fixed monocytes activated with rIFN-gamma in addition to LPS before fixation were generally more cytotoxic than those exposed to LPS alone, and those exposed to LPS were much more cytotoxic than those not exposed to LPS. Thus it is possible that high local TNF concentrations may be generated near the target cell upon direct contact between effector and target cells, and that also monocyte-associated TNF may in this way be involved in monocyte-mediated cytotoxicity.     

Subcellular localization of human monocyte interleukin 1: evidence for an inactive precursor molecule and a possible mechanism for IL 1 release

IL 1 activity, as assayed by the proliferation of responsive mouse thymocytes and a human astrocytoma cell line, was detected on the membrane of 1% paraformaldehyde-fixed activated human monocytes. Resting, unactivated monocytes did not display IL 1 activity. Maximum induction of membrane IL 1 was obtained from monocytes treated with polyclonal activators, such as LPS or Staphylococcus aureus, whereas adherence was a weak inducer of membrane IL 1. Isolated cell compartments as plasma membranes, crude lysosomes, and crude cytosol from activated human monocytes expressed significant IL 1 activity, whereas the endoplasmic reticulum showed no IL 1 activity. Exposure to trypsin of either fixed, activated human monocytes or cell compartments from unfixed monocytes, revealed biologically active IL 1 in the membrane, crude lysosome, and crude cytosol, but not in the endoplasmic reticulum. The IL 1 activity in the purified cytosol, prepared by extraction with digitonin, was considerably increased by the trypsin treatment, whereas the increase in IL 1 activity within crude lysosomes and plasma membranes was less. The cell compartments from nonactivated monocytes did not express active IL 1 and trypsin treatment revealed no active IL 1, suggesting the absence of a pool of the trypsin-sensitive form of IL 1. The data confirm the presence of membrane-bound IL 1 in activated human monocytes and indicate that an inactive precursor molecule can be found in the cytosol of such cells. Furthermore, the absence of IL 1 activity either in its active form or as the inactive precursor in the endoplasmic reticulum suggests that IL 1 is not a conventionally secreted protein. Because IL 1 was found in the cytosol as a precursor and in the lysosomal fractions in an active form, these data suggest that after the synthesis and processing of the cytosolic precursor, the 17-kda IL 1, is released via lysosomal vesicles.     

Tumor necrosis factor and IL-1 associated with plasma membranes of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both

The purpose of our study was to determine some of the mechanisms involved in macrophage-mediated lysis of tumorigenic cells. A375 human melanoma cells (A375-R) resistant to lysis mediated by TNF and IL-1 were selected from the TNF- and IL-1-sensitive A375 parental melanoma cells subsequent to continuous (2 mo) exposure to rTNF. Peripheral blood monocytes isolated by centrifugal elutriation from healthy donors were incubated with rIFN-gamma and muramyl dipeptide, with a lipoprotein derived from Escherichia coli (CG-31362) or with LPS for 24 h. These activated monocytes lysed both the A375 (monokine-sensitive) and A375-R (monokine-resistant) melanoma cells. Activated tumoricidal macrophages fixed in 2% paraformaldehyde lysed only the TNF- and IL-1-sensitive A375 cells. These fixed monocytes contained both IL-1 and TNF activities as determined by D10 cell proliferation and L929 cytolysis assays, respectively. Nearly identical results were obtained with preparations of plasma membranes from activated human monocytes. Anti-IL-1 and/or anti-TNF sera neutralized the cytolysis of tumor cells mediated by free monokines, by fixed monocytes, or by plasma membrane preparations. In contrast, anti-TNF and/or anti-IL-1 sera did not inhibit tumor cell lysis by viable activated monocytes. We conclude that IL-1 and TNF molecules associated with the plasma membranes of activated monocytes mediate lysis of susceptible target cells. However, because activated monocytes lysed IL-1-and TNF-resistant target cells, molecules other than these monokines must also be involved in the antitumor activity of monocytes.     

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.     

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.     

Effects of botulinum toxin type D on secretion of tumor necrosis factor from human monocytes.

Botulinum toxins are potent neurotoxins which block the release of neurotransmitters. The effects of these toxins on hematopoietic cells, however, are unknown. Monocytes secrete a variety of polypeptide growth factors, including tumor necrosis factor (TNF). In the study reported here, the effects of botulinum toxin type D on the secretion of TNF from human monocytes were examined. The results demonstrate that botulinum toxin type D inhibits the release of TNF from monocytes activated by lipopolysaccharide (LPS) but not by 12-O-tetradecanoylphorbol-13-acetate. Botulinum toxin type D had no detectable effect on intracellular TNF levels in LPS-treated monocytes, indicating that the effects of this toxin involve the secretory process. This inhibitory effect of botulinum toxin type D on TNF secretion from LPS-treated monocytes was partially reversed by treatment with 12-O-tetradecanoylphorbol-13-acetate or introduction of guanosine 5'-[gamma-thio]triphosphate into these cells. The results demonstrate that TNF secretion is regulated by at least two distinct guanine nucleotide-binding proteins, one responsible for the activation of phospholipase C and another which acts as a substrate for botulinum toxin type D. ADP-ribosylation of monocyte membranes by botulinum toxin type D demonstrated the presence of three substrates with Mrs of 45,000, 21,000, and 17,000. While the role of these substrates in exocytosis is unknown, the results suggest that the Mr 21,000 substrate is involved in a process other than TNF secretion.