Biomedical subjects

K L Bost

Publications and source records attributed to K L Bost.

At least 37 records · Page 2Linked to original sources

PubMed1999

Staphylococcus aureus infection of mouse or human osteoblasts induces high levels of interleukin-6 and interleukin-12 production.

Staphylococcus aureus is the principal causative agent of the inflammatory bone disease osteomyelitis. Unfortunately, the pathogenesis of this often chronic infection is poorly understood and is complicated by the recent observation that bone-forming osteoblasts can harbor S. aureus. Such an infection presents a significant challenge for the host immune response, because osteoblasts are not known to initiate protective cell-mediated immune responses. Cultured mouse and human osteoblasts infected with S. aureus were found to express high levels of interleukin (IL)-6 and IL-12p75, on the basis of complementary investigations demonstrating both S. aureus-induced up-regulation of expression of IL-6 and IL-12p40 mRNA and secretion of IL-6 and IL-12p75 by these cells. Additionally, a quantitative bioassay demonstrated that IL-12p75 secreted after infection was biologically active. These studies are the first to demonstrate induced IL-12p75 expression by osteoblasts and suggest a previously unrecognized role for osteoblasts in initiating immune responses after S. aureus infection.

Animals↗

PubMed1999

Limited interleukin-18 response in Salmonella-infected murine macrophages and in Salmonella-infected mice.

Optimal immune responses against an intracellular bacterial pathogen, such as Salmonella, involve the production of gamma interferon (IFN-gamma), which activates macrophages. It has recently been suggested that, interleukin-18 (IL-18), in addition to IL-12, contributes to the induction of IFN-gamma following infection. Given this hypothesis, an optimal host immune response against intracellular bacterial pathogens would include the induction of IL-18 secretion by macrophages due to Salmonella infection. We questioned whether Salmonella could induce macrophages to upregulate their expression of IL-18 mRNA and secretion of IL-18. With cultures of murine macrophages, we were surprised to find that infection by wild-type Salmonella dublin resulted in decreased expression of IL-18 mRNA and IL-18 secretion rather than an increase. Reduction of macrophage-derived IL-18 expression by wild-type Salmonella occurred early in the response, suggesting a direct effect. Furthermore, mice orally inoculated with wild-type Salmonella were shown to have reduced IL-18 mRNA expression at mucosal sites within hours postinoculation. Together these studies demonstrate Salmonella-induced reductions in IL-18 expression, suggesting that this intracellular pathogen may be capable of limiting a potentially protective immune response.

Amino Acid Sequence↗

PubMed1999

CD40-CD40 ligand interactions augment survival of normal mice, but not CD40 ligand knockout mice, challenged orally with Salmonella dublin.

Interactions between CD40 expressed on macrophages and CD40 ligand expressed on T lymphocytes can be an important signal for optimal macrophage activation. Previous studies have demonstrated that the optimal response against certain intracellular pathogens (e.g., Crytosporidium and Leishmania spp.) by macrophages requires CD40-CD40 ligand interactions. However, this finding is not universal, since two recent reports utilizing CD40 knockout mice have shown no such contribution to the protective immune response against Mycobacterium tuberculosis or Histoplasma capsulatum. We demonstrate here that CD40-CD40 ligand interactions are significant events in the protective response against the intracellular pathogen Salmonella dublin in normal mice but not for animals genetically deficient in CD40 ligand expression. Treating BALB/c mice exogenously with a CD40 agonist (i.e., soluble trimeric CD40 ligand) increased resistance against a lethal, orally administered dose of S. dublin. Conversely, in vivo administration of a monoclonal antibody against CD40 ligand to block endogenous CD40-CD40 ligand interactions resulted in a decreased resistance to salmonellosis. In contrast, CD40 ligand knockout mice demonstrated no increased susceptibility to salmonellosis. In vitro treatment of Salmonella-infected macrophages from BALB/c mice with soluble trimeric CD40 ligand resulted in an elevated production of interleukin 12p70 by these cells, suggesting a mechanism whereby CD40-CD40 ligand interactions might enhance protective immune responses to this pathogen. Taken together, these studies strongly suggest that CD40-CD40 ligand interactions in normal mice play an important protective role in immune responses against the gram-negative, intracellular pathogen S. dublin.

Animals↗

PubMed1998

Substance P diminishes lipopolysaccharide and interferon-gamma-induced TGF-beta 1 production by cultured murine macrophages.

Recent evidence has demonstrated the importance of substance P and its receptor in macrophage-mediated inflammatory responses. While previous studies have shown that substance P can augment proinflammatory monokine production, little is known about the effects of this neuropeptide on the production of monokines that might limit inflammation. In the present study we have investigated the effect of substance P treatment on the production of transforming growth factor-beta 1 (TGF-beta 1) in cultured murine macrophages. We report that, while substance P agonist alone elicited increases in TGF-beta 1 mRNA expression and modest increases in TGF-beta 1 secretion, substance P dramatically diminished LPS- or IFN-gamma-induced TGF-beta 1 production. These results suggest a previously unrecognized mechanism where substance P may act as a proinflammatory mediator by limiting the production of excessive levels of TGF-beta 1 by LPS- or IFN-gamma-activated macrophages.

Animals↗

PubMed1998

Differential kinetics for induction of interleukin-6 mRNA expression in murine peritoneal macrophages: evidence for calcium-dependent and independent-signalling pathways.

It is presently unclear what role elevations in intracellular calcium concentration ([Ca2+]i) play in the control of monokine secretion, or whether such alterations underlie the ability of physiologic stimuli to induce production of these important signalling molecules. To address these issues, we have performed experiments in murine peritoneal macrophages to determine whether lipopolysaccharide (LPS) or interferon gamma (IFN-gamma) initiate production of the proinflammatory monokine interleukin 6 (IL-6) concomitant with elevations in [Ca2+]i and with kinetics similar to that seen with known Ca2+ mobilizing agents. Alterations in [Ca2+]i after treatment with LPS, IFN-gamma, platelet activating factor (PAF), or thapsigargin were measured by fluorimetric methods. These effects were compared with the ability of each to induce IL-6 mRNA expression as measured by semiquantitative reverse-transcribed polymerase chain reactions. We report that neither LPS nor IFN-gamma elicited detectable elevations in [Ca2+]i but that both up-regulated expression of IL-6 mRNA expression within 60 min. In contrast, experiments using either thapsigargin or PAF showed rapid and dramatic elevations in [Ca2+]i with marked increases in IL-6 mRNA expression, as quickly as 15 min after initial exposure. Elevations in mRNA encoding IL-6 by thapsigargin and PAF were found to occur in a dose-dependent manner, mirroring their ability to elicit elevations in [Ca2+]i. These data demonstrate that LPS and IFN-gamma induce IL-6 message expression by means of Ca2+-independent signalling pathways. Furthermore, Ca2+-mobilizing agents that evoke monokine message expression do so far more rapidly than do LPS or IFN-gamma. Taken in concert, these data are consistent with the hypothesis that multiple signalling pathways exist by which production of proinflammatory monokines are initiated.

Animals↗

PubMed1998

Cytokine involvement in immunomodulatory activity affected by Candida albicans mannan.

Candida albicans mannoprotein (MAN) administered intravenously to mice stimulates the production of splenic CD8+ effector cells which downregulate delayed hypersensitivity (DH) in immunized mice. Cytokine involvement in the induction and/or elicitation of downregulation was studied by (i) examining murine splenocytes qualitatively for mRNA for interleukin-2 (IL-2), IL-4, IL-10, IL-12p40, and gamma interferon (IFN-gamma), (ii) quantitating splenocyte mRNA for IL-12p40 by quantitative-competitive reverse transcriptase-mediated PCR, and (iii) measuring serum levels of IL-12p40 and IL-12p70 by capture enzyme-linked immunosorbent assay, each performed at selected intervals over 96 h after giving MAN. Further, the effect of in vivo administration of anti-IL-4 on the induction and elicitation of MAN-specific DH in MAN-treated mice was measured. Expression of IL-12p40 mRNA in the spleen was reduced to near 0 during the first 24 h but rebounded thereafter. Transcripts for IL-10 were present throughout the 96-h period, whereas those for IL-4 and IFN-gamma were either weak or undetectable prior to 24 to 48 h. In vivo administration of anti-IL-4 partially abrogated the downregulatory effect of MAN only when given at the time of MAN administration. Serum levels of IL-12p40, but not IL-12p70, were increased by 24 h and maximal at 48 h. The antagonistic effect of IL-12p40 could contribute to the mechanism(s) for downregulation of DH. Moreover, IL-10, IL-4, and/or IFN-gamma, interacting with MAN-activated cells in the absence of biologically active IL-12, may induce the production of CD8+ downregulatory effector cells. Partial abrogation of downregulatory activity in animals treated with anti-IL-4 at the time of induction of such activity lends support to this hypothesis.

Animals↗

PubMed1998

Limitations for purification of murine interleukin-18 when expressed as a fusion protein containing the FLAG peptide.

As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affinity columns could be performed. While significant amounts of recombinant IL-18 were present in E. coli lysates, only a small portion of this material could be recovered on immunoaffinity columns conjugated with an anti-FLAG antibody. Surprisingly, the majority of recombinant IL-18 present in E. coli (strain JM83) bacterial lysates did not contain the FLAG peptide and therefore did not bind to immunoaffinity columns conjugated with an anti-FLAG antibody. However, we found that the BL21 strain of E. coli, which has reduced endogenous protease activity, could express the majority of recombinant IL-18 as the fusion protein, FLAG-IL-18. Taken together, these studies show that it is necessary to consider whether protease sites formed at the FLAG-protein junction can be easily cleaved by the bacterial strain used to express the fusion protein.

Amino Acid Sequence↗

PubMed1997

Substance P-induced IL-12 production by murine macrophages.

Previous investigations in our laboratory have suggested that substance P (NK-1) receptor expression by macrophages contributes to the resistance against the intracellular bacterial pathogen, Salmonella. To investigate possible mechanisms for such resistance, macrophages were cultured with varying concentrations of a substance P agonist to investigate the ability of this neuropeptide to augment IL-12 expression. The substance P agonist was a potent inducer of both IL-12p35 and IL-12p40 mRNA expression in cultured macrophages. The kinetics of this response were maximal within 6 h and could be observed with concentrations of substance P agonist as low as 0.1 nM. The nonpeptide, substance P receptor antagonist, CP96-345, significantly blocked agonist-induced IL-12 mRNA expression, further demonstrating that this effect was mediated through an NK-1 receptor. Substance P agonist alone could stimulate substantial secretion of IL-12p40, but not IL-12p70, by cultured macrophages. Thus, the substance P agonist had the ability to augment IL-12p35 and IL-12p40 mRNA expression, but not to increase IL-12p70 secretion. Like IFN-gamma, we found that substance P could combine with LPS to significantly augment the secretion of bioactive IL-12p70. The costimulatory effects of substance P agonist plus LPS on IL-12 mRNA expression were additive; however, this combination resulted in synergistic secretion of IL-12p70 by macrophages. Together, these results demonstrate the ability of NK-1 receptors to signal IL-12 production by macrophages and suggest mechanisms for substance P-induced modulation of cellular immunity.

Adjuvants, Immunologic↗

PubMed1997

Intracellular Salmonella dublin induces substantial secretion of the 40-kilodalton subunit of interleukin-12 (IL-12) but minimal secretion of IL-12 as a 70-kilodalton protein in murine macrophages.

The induction by intracellular pathogens of interleukin-12 (IL-12) secretion is of particular importance since this cytokine has been shown to be necessary for optimal cell-mediated immune responses. Several recent investigations have suggested that cultured macrophages are a significant source of IL-12 following intracellular infection with pathogens such as Salmonella spp. In an effort to critically evaluate the magnitude of the IL-12 response in cultured macrophages following interaction with Salmonella dublin, enzyme-linked immunosorbent assays specific for the 40- and 70-kDa subunits of IL-12 (IL-12p40 and IL-12p70) and a sensitive bioassay for IL-12p70 were used. Using BALB/c macrophages, S. dublin at various challenge doses was a potent inducer of IL-12p40 secretion (>6,000 pg/10(7) macrophages). However when secretion of IL-12p70 was evaluated, S. dublin did not induce comparable IL-12p70 production (<80 pg/10(7) macrophages) at any time, despite varying the challenge dose of Salmonella. The limited ability of BALB/c (Ity(s)) macrophages to secrete IL-12p70 in response to Salmonella was not a strain-specific phenomenon since similar results were demonstrated for macrophages isolated from CBA/J (Ity(r)) and C3H/HeJ (lipopolysaccharide [LPS]-hyporesponsive) mice. While intracellular infection with Salmonella was not a potent stimulus for IL-12p70 secretion in these mouse strains, macrophages from these mice responded significantly to a stimulus of gamma interferon plus LPS. Taken together these results demonstrate a limited capacity for intracellular Salmonella to stimulate murine macrophage secretion of IL-12p70, despite being a significant stimulus for IL-12p40 secretion. Furthermore, our results suggest that Salmonella-induced IL-12p40 secretion by macrophages is not solely an LPS-mediated event.

Animals↗

PubMed1997

Reverse transcriptase-polymerase chain reaction amplification and partial sequence of T helper 1- and T helper 2-type lymphokine genes from the owl monkey (Aotus trivirgatus).

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify selected lymphokine mRNAs from phytohemagglutinin-activated leukocytes of the owl monkey (Aotus trivirgatus). Interleukin-2 (IL-2), IL-4, IL-13, and interferon-gamma were selected as lymphokine mRNAs of interest, since expression of these cytokines helps define the type of T helper lymphocyte response (i.e., TH1 versus TH2). Because sequences for these lymphokine genes were not available for the owl monkey, multiple PCR primers for each lymphokine gene were designed based on published human sequences. Various PCR primer pairs were then used in the RT-PCR to determine the conditions for optimal amplification of each owl monkey cytokine mRNA. In addition, each PCR primer pair was compared for the ability to amplify lymphokine mRNAs from other primate species, including African green (Cercopithecus aethiops), squirrel (Saimiri sciureus), and rhesus (Macaca mulatta) monkeys. The specificity and sensitivity of optimal primer pair was also demonstrated by amplification of as little as 10 fg of each lymphokine gene in a background of 300 ng of irrelevant cDNA. Finally, partial sequences of owl monkey coding regions for IL-2, IL-13, and interferon-gamma were determined and compared for homology with their human counterparts. Together, these studies define specific and sensitive conditions for detection of lymphokine mRNA expression in the owl monkey and provide partial sequence information of the coding region for these lymphokines. This investigation should provide molecular probes to investigate the immune response against malaria and the effectiveness of malaria vaccines in the owl monkey that models this human disease.

Amino Acid Sequence↗

PubMed1996

Increased susceptibility of mice to Salmonella infection following in vivo treatment with the substance P antagonist, spantide II.

Successful resolution of salmonellosis in naive mice depends in large part upon IL-12-induced IFN-gamma production to eliminate this intracellular pathogen of macrophages. In the present study we questioned the contribution that expression of substance P receptors makes to the protective response following oral inoculation with a lethal dose of Salmonella. Such a relationship was suggested when oral inoculation with Salmonella induced rapid and dramatic increases in substance P receptor mRNA expression within Peyer's patches and mesenteric lymph nodes and subsequently in the spleen. The importance of substance P receptor expression in vivo was further suggested by pretreatment of mice with the substance P antagonist, spantide II, before oral inoculation with Salmonella. Mice pretreated with spantide II and then orally inoculated developed advanced salmonellosis and had significantly reduced survival rates compared with mice pretreated with a control peptide. Treatment with spantide II significantly reduced early Salmonella-induced IL-12p4O and IFN-gamma mRNA expression at mucosal sites, suggesting a mechanism for the reduced ability of spantide II-treated mice to resist this pathogen. Increased susceptibility to salmonellosis was not due to 1) spantide II-induced alterations in the uptake of this pathogen from the gut, 2) global spantide II-mediated immune suppression, or 3) nonsubstance P receptor-mediated effects of spantide II on macrophages. The ability of Salmonella to induce substance P receptor expression on cultured macrophages suggested that one mechanism for resistance against this intracellular pathogen might be a direct effect of substance P on this cell population.

Administration, Oral↗

PubMed1996

In vivo treatment with anti-interleukin-13 antibodies significantly reduces the humoral immune response against an oral immunogen in mice.

Interleukin-13 (IL-13) is a cytokine which significantly enhances the proliferation and differentiation of B lymphocytes. We therefore evaluated its role in the formation of a humoral immune response in vivo. Upon oral immunization with the B subunit of Escherichia coli heat-labile enterotoxin (LT-B), rapid up-regulation of IL-13 mRNA expression in the mesenteric lymph nodes of LT-B intubated mice occurred. This result suggested that IL-13 might be involved in the formation of a mucosal antibody response against LT-B if this cytokine was in fact secreted. To test this possibility, the coding region for murine IL-13 was cloned into the pFLAG-1 expression vector. Recombinant murine IL-13 was purified from bacterial lysates and used as an immunogen to produce polyclonal anti-IL-13 antibodies. Groups of BALB/c mice treated in vivo with anti-IL-13 antibody 2 days before and on the day of oral immunization with LT-B had significantly reduced intestinal IgA and serum IgG and IgA anti-LT-B antibody responses when compared to mice treated with control antibody. Furthermore, groups of mice primed with LT-B and then treated with anti-IL-13 antibody prior to oral immunization with a second dose of LT-B also had significantly reduced intestinal IgA and serum IgG and IgA anti-LT-B antibody titres compared to controls. In vitro LT-B restimulation experiments using splenic mononuclear leucocytes isolated from LT-B primed mice treated with anti-IL-13 antibody demonstrated decreased expression of IL-4 and IL-13 mRNA and decreased IL-4 secretion when compared to controls. Together these results demonstrate an important role for IL-13 in the formation of a humoral immune response at mucosal surfaces.

Administration, Oral↗

PubMed1996

Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin.

Escherichia coli labile toxin (LT) was assessed as mucosal immunogen and as adjuvant for tetanus toxoid (TT) in mice. After oral administration of LT, C57BL/6 (H-2b) and BALB/c(H-2d) mice were high mucosal and serum antibody responders, while C3H/HeN (H-2k) mice were low responders. High responders exhibited mainly serum IgG (including IgG1, IgG2a, and IgG2b), as well as IgM and IgA, while mucosal responses were IgA. Analysis of LT-B-specific CD4+ T helper (Th) cells from Peyer's patches (PP) or from spleen revealed a mixed Th1 (interferon-gamma) and Th2 (interleukin-4 and -5) cell pattern. Oral LT given with TT induced TT-specific response patterns identical to LT-B. Analysis of mRNA from TT-specific PP CD4+ Th cells also revealed a mixed Th1- and Th2- type response. Thus, antibody response profiles induced by LT are regulated by both CD4+ Th1 and Th2 cell types.

Adjuvants, Immunologic↗

PubMed1996

Differential production of interleukin-12 mRNA by murine macrophages in response to viable or killed Salmonella spp.

The use of attenuated Salmonella spp. as live oral vaccine carriers fo r foreign antigens has been extensively studied. We have shown that appropriately prepared nonviable organisms are as effective as viable organisms in eliciting humoral immune responses against a foreign antigen delivered by these vectors. It is not clear how strain viability affects the development of a cell-mediated immune response. In the present study, we demonstrate that BALB/c mice orally immunized with viable attenuated Salmonella spp. were protected against subsequent challenge while animals immunized with killed organisms were not. Protection was correlated with increased production of interleukin-12 (IL-12) p40 mRNA in the Peyer's patches within hours of oral administration. Peritoneal macrophages from lipopolysaccharide (LPS)-responsive and LPS-unresponsive mice were also examined for production of IL-12 p40 mRNA following exposure to the viable or killed attenuated Salmonella carrier. There was dramatic upregulation of IL-12 p40 mRNA following exposure of macrophages to either viable or killed organisms. By 4 h postexposure, viable organisms had induced a 27-fold increase in IL-12 p40 mRNA levels while killed organisms had induced a 9-fold increase in IL-12 p40 mRNA levels. This was observed in macrophages isolated from both LPS-responsive and unresponsive mice. The higher levels of IL-12 induced by viable Salmonella spp. may result in the development of a Th1 response and cell mediated immunity, while the lower levels of IL-12 induced by killed Salmonella spp. may not be sufficient to promote a Th1 response.

Animals↗

PubMed1996

Epitope maps of the Escherichia coli heat-labile toxin B subunit for development of a synthetic oral vaccine.

Linear B- and T-cell epitopes spanning all 103 amino acids of the Escherichia coli heat-labile toxin B subunit (LT-B) were assessed in mice orally immunized with native LT or with recombinant Salmonella enteritidis expressing LT-B. Oral administration of native LT induced mucosal immunoglobulin A (IgA) antibodies reactive with an epitope at residues 85 to 91, while IgA induced by recombinant Salmonella LT-B reacted with an epitope at residues 36 to 44. Serum IgG anti-LT-B antibodies from mice orally immunized with either LT or with recombinant Salmonella LT-B were directed to both epitopes. A single T-cell epitope spanning residues 34 to 42 was identified by T-cell proliferative and cytokine responses. When a 20-mer peptide (residues 26 to 45) with B- and T-cell epitopes was given orally to BALB/c (H-2(d)) and B10 congenic (I-A(d), I-A(b), and I-A(k)) mice, significant fecal IgA and serum IgG anti-LT-B antibodies were induced. The peptide also induced LT-B-specific T-cell proliferative responses in these mice. Orally administered LT-B peptide (residues 26 to 45) induced a cytokine profile indicative of both T helper 1- and 2-type cells. The remarkable immunogenicity of this 20-mer peptide makes it a candidate for a vaccine to protect against enterotoxigenic E. coli.

Administration, Oral↗

PubMed1996

Endogenous and exogenous interleukin-12 augment the protective immune response in mice orally challenged with Salmonella dublin.

Following oral challenge with Salmonella dublin, we observed significant increases in interleukin-12 (IL-12) protein expression in the mesenteric lymph nodes. The importance of this endogenous cytokine production in the immune response against S. dublin was demonstrated by in vivo depletion of IL-12 with an anti-IL-12 monoclonal antibody prior to oral S. dublin challenge. Mice pretreated with anti-IL-12 antibody had increased salmonellosis and reduced survival times compared with mice receiving control antibody. Furthermore, administration of exogenous murine recombinant IL-12 dramatically increased survival times of mice challenged orally with S. dublin. Together, these results demonstrate that endogenous and exogenous IL-12 significantly augment the mucosal immune response against the intracellular pathogen S. dublin.

Administration, Oral↗

PubMed1996

Mechanisms that contribute to the development of lymphoid malignancies: roles for genetic alterations and cytokine production.

Recent studies have defined genetic alterations commonly associated with transformed lymphocytes. This review suggests roles for these alterations in the development of lymphoid neoplasms. Damage to the genes encoding proteins that function in intracellular signaling, transcription, or regulation of the cell cycle has been identified and linked at varying degrees to the progression of certain lymphoid malignancies. An understanding of the mechanistic consequences following such genetic alterations is essential to an understanding of the development of these lymphoid neoplasms. In contrast, it is also becoming clear that the dysregulated expression of proteins that are not genetically altered can also contribute to the progression of lymphoid malignancies. One such example is the excessive expression of "normal" lymphokines of cytokines which accompanies many lymphoproliferative diseases. The dysregulated expression of cytokines during malignancy can result in the augmentation of growth of transformed lymphocytes, as well as an alteration of the anti-tumor immune response. The latter mechanism is especially important because evasion of the impending immune response is a prerequisite for the progression of lymphoproliferative diseases. Taken together, this review supports the notion that the development of lymphoid malignancies is multifactorial, involving genetic alterations as well as dysregulated cytokine expression.

Animals↗

PubMed1995

Thapsigargin and cyclopiazonic acid initiate rapid and dramatic increases of IL-6 mRNA expression and IL-6 secretion in murine peritoneal macrophages.

Two different inhibitors of endosomal calcium ATPase activity, cyclopiazonic acid and thapsigargin, were shown to release a common intracellular calcium pool in normal, murine macrophages. Furthermore, the release of this pool was accompanied by increased calcium uptake from the extracellular medium. The activity of these inhibitors was linked to an important biologic response, because both cyclopiazonic acid and thapsigargin induced rapid and dramatic increases in IL-6 mRNA expression and secretion. Compared with control cultures, macrophages treated with these inhibitors increased IL-6 mRNA expression approximately 10-fold by 15 min and approximately 20-fold by 2 h, as determined using quantitative competitive-reverse transcribed-PCRs. The increased mRNA expression was coupled to translation and secretion of this monokine since cyclopiazonic acid and thapsigargin induced significant increases in IL-6 secretion as early as 2 h, and up to approximately 70-fold increases by 20 h, when compared with control cultures. Taken together, these results demonstrate that both cyclopiazonic acid and thapsigargin generate potent intracellular signals that initiate rapid and dramatic production of IL-6. Both thapsigargin and cyclopiazonic acid increased IL-6 mRNA expression at 15 min in the absence of Ca2+ influx from the extracellular medium. These results suggest that events associated with endosomal Ca(2+)-ATPase inhibition contribute to the activation of normal macrophages as defined by increased monokine secretion.

Animals↗