Biomedical subjects

D C Morrison

Publications and source records attributed to D C Morrison.

At least 19 recordsLinked to original sources

PubMed2001

Bacterial DNA and lipopolysaccharide induce synergistic production of TNF-alpha through a post-transcriptional mechanism.

LPS is well recognized for its potent capacity to activate mouse macrophages to produce TNF-alpha, an important inflammatory mediator in bacterial infection-related diseases such as septic shock. We demonstrate here that while inducing only low levels of TNF-alpha alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce TNF-alpha in the RAW 264.7 macrophage-like cell line. The bacterial DNA effects are mimicked by synthetic CpG-containing oligodeoxynucleotides, but not non-CpG-containing oligodeoxynucleotides. Pretreatment of macrophages with either DNA for 2-8 h inhibits macrophage TNF-alpha production in responses to DNA/LPS. However, when pretreatment was extended to 24 h, DNA/LPS synergy on TNF-alpha is further enhanced. RT-PCR analysis indicates that mRNA levels of the TNF-alpha gene, however, are not synergistically induced by bacterial DNA and LPS. Analyses of the half-life of TNF-alpha mRNA indicate that TNF-alpha message has a longer half-life in bacterial DNA- and LPS-treated macrophages than that in bacterial DNA- or LPS-treated macrophages. These findings indicate that the temporally controlled, synergistic induction of TNF-alpha by bacterial DNA and LPS is not mediated at the transcriptional level. Instead, this synergy may occur via a post-transcriptional mechanism.

Adjuvants, Immunologic↗

PubMed2001

Interleukin-6 production by endothelial cells via stimulation of protease-activated receptors is amplified by endotoxin and tumor necrosis factor-alpha.

Human endothelial cells respond to extracellular proteases, endotoxin (lipopolysaccharide, LPS), and inflammatory cytokines. Endothelial cells express several protease-activated receptors (PAR), including the thrombin-activated receptors PAR-1 and PAR-3 and a thrombin-independent, protease-activated receptor, PAR-2. To examine the potential cooperation between PAR and inflammatory stimuli, we investigated the effects of the PAR-1 agonist peptide Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and PAR-2 agonist peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro with SFLLRN or SLIGKV in the presence and absence of LPS or tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) levels in the culture supernatants were assayed. Both SFLLRN and SLIGKV induced detectable levels of IL-6 production in a dose-dependent fashion, with the PAR-1 receptor agonist being more potent. In the presence of all stimulatory concentrations of LPS or TNF-alpha tested, both peptides were found to further enhance IL-6 production. The effects of SFLLRN and SLIGKV were specific, as related peptides with identical amino acid compositions, but lacking in consensus sequences, were biologically inactive either alone or in the presence of LPS. Both the direct and the amplifying effects of PAR agonist peptides on IL-6 production were pertussis toxin sensitive and caused an increase in the intracellular levels of calcium, implicating G-proteins and calcium mobilization in these pathways. Furthermore, the amplifying effect of LPS or TNF-alpha on PAR-mediated cytokine production was associated with corresponding increases in nuclear NF-kappaB proteins. The results demonstrate significant potentiation of PAR-induced signaling by LPS and TNF-alpha and indicate the potential cooperation of proteases and inflammatory stimuli in amplifying vascular inflammation.

Calcium↗

PubMed2001

Commercial preparations of lipoteichoic acid contain endotoxin that contributes to activation of mouse macrophages in vitro.

Lipoteichoic acids (LTA), cell wall components of gram-positive bacteria, have been reported to induce various inflammatory mediators and to play a key role in gram-positive-microbe-mediated septic shock. In a large number of these studies, investigators used commercially available LTA purified from a variety of gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, and Streptococcus sanguis. We report here that, although these commercially available LTA could be readily shown to stimulate production of nitric oxide (NO) in RAW 264.7 mouse macrophages, the activity was dramatically inhibited by polymyxin B, a relatively specific inhibitor of endotoxin biological activity. One-step purification of the commercially available S. aureus LTA using hydrophobic interaction chromatography resulted in two well-separated peak fractions, one highly enriched for LTA and a second highly enriched for endotoxin. The LTA-enriched fractions did not induce production of NO in RAW 264.7 macrophages, although they caused a dose-dependent induction of NO in the presence of low concentrations of gamma interferon (IFN-gamma) (which by itself induced little NO), regardless of the presence of polymyxin B. In contrast, the endotoxin-enriched fractions by themselves inhibited in high levels of NO in RAW 264.7 macrophages but activity was almost completely inhibited in the presence of polymyxin B. Consistent with these findings, our data also indicate that commercial LTA preparations from S. aureus, B. subtilis, and S. sanguis were not able to induce NO from lipopolysaccharide-hyporesponsive C3H/HeJ mouse peritoneal macrophages, but in the presence of IFN-gamma, these LTA preparations were able to induce relatively high levels of NO from C3H/HeJ macrophages. These results indicate that commercially available LTA can contain contaminating and potentially significant levels of endotoxin that can be expected to contribute to the putative macrophage-stimulating effects of LTA as assessed by NO production. The fact that the purified LTA, by itself, was not able to induce significant levels of NO secretion in RAW 264.7 macrophages supports the conclusion that caution in attributing high-level biological activity to this microbial cell wall constituent should be exercised.

Animals↗

PubMed2001

Lipopolyamines as a therapeutic strategy in experimental Gram-negative bacterial sepsis.

Lipopolyamines are a class of polycationic amphiphilic compounds that have been shown to bind with high affinity to polyanionic macromolecules, including both DNA and bacterial lipopoly-saccharide (LPS). One of these compounds, termed DOSPER (1,3-di-oleoyloxy-2-(6-carboxyl-spermyl)- propylamide), is non-cytotoxic and has been shown to inhibit LPS-mediated cytokine release and lethality in endotoxin challenge models. In the study reported here, the activity of DOSPER was tested in neutropenic rats with invasive Gram-negative bacteremia caused by Pseudomonas aeruginosa. DOSPER alone was ineffective (0/8) at influencing mortality, but provided a significant survival advantage if administered in combination with a bactericidal antibiotic, ceftazidime (10/12; P<0.05). Ceftazidime alone was partially protective (6/12) while the control group had no survivors (0/8). DOSPER administration markedly reduced circulating endotoxin levels (P<0.01) and interleukin-6 levels (P<0.05) but had no significant effect on bacteremia and bacterial concentrations of P. aeruginosa in liver or spleen tissue. Lipopolyamines may be potentially valuable as a therapeutic adjunct in treatment of Gram-negative bacterial sepsis.

Animals↗

PubMed2001

Alteration of surfactant proteins A and D in bronchoalveolar lavage fluid of Pneumocystis carinii pneumonia.

OBJECTIVE: To understand the interaction between surfactant proteins and pneumocystis carinii pneumonia (PCP), and the impact of corticosteriods on surfactant proteins. METHODS: We established rat models of PCP and bacterial pneumonia induced by subcutaneous injection of 25 mg cortisone acetate. At 8-12 wk, the bronchoalveolar lavage fluid (BALF) of rats was collected. Total nucleated cells of BALF were counted and differentiated, and the concentrations of surfactant protein A (SP-A) and surfactant protein D (SP-D) were measured by immunoblotting assay. The rats were divided into three immunosuppressive groups and a normal control group. Group I, normal control (n = 6), consisted of healthy SD rats; group II, negative control (n = 6), consisted of rats with cortisone acetate injection for over 8 wk without lung infection; group III, bacterial pneumonia (n = 11), rats were injected with cortisone acetate over 8 wk that resulted in bacterial pneumonia without other pathogens isolated; and group IV, PCP (n = 14), rats with injected cortisone acetate for 8-12 wk and developed PCP without other pathogens isolated. RESULTS: Our results indicated that the total cell count in BALF in the negative control group was lower than that in the normal control group (P < 0.001). During PCP infection, the total cell count and the percentage of polymorphonuclearcytes (PMNs) in BALF were significantly increased (P < 0.01), but were lower than those in the bacterial pneumonia group. The concentration of SP-A of BALF in PCP (45.1 +/- 22.1 micrograms/ml) was significantly increased in comparison with that in the negative control (16.2 +/- 9.9 micrograms/ml, P < 0.05) and bacterial pneumonia groups (6.2 +/- 5.6 micrograms/ml, P < 0.001). We also found that the relative content of SP-D was significantly higher in PCP (24,249 +/- 4780 grey values) than that in the negative control (13,384 +/- 2887 grey values, P < 0.001) and that in bacterial pneumonia (11,989 +/- 2750 grey values, P < 0.001). SP-A and SP-D were also higher in the moderate to heavy group of PCP than those seen in the mild group (P < 0.01, P < 0.001). SP-A and SP-D were higher in the negative control group than those in the normal control group, but there was no significant difference between the 2 groups. CONCLUSION: These results suggest that the concentrations of SP-A and SP-D in BALF are increased by pneumocystis carinii specific stimulation, but the alteration is not related to the corticosteriod usage.

Animals↗

PubMed2000

What are the microbial components implicated in the pathogenesis of sepsis? Report on a symposium.

Despite considerable efforts in the past quarter century to improve therapy for sepsis, mortality rates remain unacceptably high. Microbe-derived constituents can induce the host to produce many mediators that can contribute to immune dysregulation, tissue damage, and death. Although endotoxin-mediated events are clearly important in gram-negative infections, gram-positive bacteria can also play a dominant role. Understanding the interplay of microbial constituents and host immune or inflammatory responses prompted a meeting at Rockefeller University in May 1998. Participants discussed the relative merits of a "2-hit" hypothesis to explain the course of lethal septic shock and a "multihit" synergistic threshold hypothesis. Recommendations include the following: (1) developing animal models that closely mimic human sepsis; (2) further investigating antibiotic effects on bacteria; (3) assessing the relationships between endotoxin, prokaryotic DNA, and peptidoglycan (i.e., independent, additive, or synergistic) in inducing host responses; and (4) developing new strategies to improve outcomes. Studies are needed to better define which and how different microbial constituents lead to sepsis and to provide critical leads for therapeutic intervention.

Amino Acid Sequence↗

PubMed2000

Prenatally drug-exposed toddlers: cognitive and social development.

In a replication of an earlier study, 122 prenatally drug-exposed toddlers were assessed on four cognitive scales and rated by their caregivers on socialization, communication, and daily living skills. A significant number of the sample performed below age expectancy on the cognitive scales and on at least two of the three domains rated by caregivers.

Child, Preschool↗

PubMed2000

Comparable endotoxic properties of lipopolysaccharides are manifest in diverse clinical isolates of gram-negative bacteria.

In general there is a poor correlation between serum lipopolysaccharide (LPS; the biologically active constituent of endotoxin) levels and mortality in septic patients. The objective of this study was to determine if chemical, structural, or biological differences among LPS from different clinical isolates of gram-negative bacteria might explain this discrepancy. LPS preparations were made using the hot phenol-water extraction method from eight clinical isolates of gram-negative bacteria. As a percentage of the total weight of the LPS, the phosphate content ranged from 3.0 to 13.8% (average, 6.7 +/- 3.6%), and the 2-keto-3-deoxyoctonate content ranged from 1.9 to 27.4% (average, 8.9 +/- 8.5%). These values were not dissimilar to those obtained for a reference endotoxin. In a standard measure of LPS activity, the Limulus amoebocyte lysate assay, there was approximately a twofold difference between the least and most active preparations. The two preparations with the greatest difference in their ability to elicit the secretion of tumor necrosis factor alpha from a mouse peritoneal macrophage cell line were similar in lethality when administered to mice sensitized to the effects of LPS by D(+)-galactosamine. These relatively minor differences in LPS activity seem unlikely to explain the generally observed discrepancy between serum endotoxin levels and mortality in patients with gram-negative sepsis.

Animals↗

PubMed2000

Differential host inflammatory responses to viable versus antibiotic-killed bacteria in experimental microbial sepsis.

Staphylococcus aureus killed during imipenem or ceftazidime chemotherapy in mice elicited an early release of tumor necrosis factor alpha (TNF-alpha) into the systemic circulation. This response was coincident in time with an increase in leukocyte-endothelium adhesive interactions in the microvasculature. Equivalent responses were not observed without the antibiotic treatment (imipenem or ceftazidime). Protective efficacy of the same antibiotic treatment was markedly diminished in D-galactosamine-treated mice compared to controls; e.g., it dropped from 2,000-fold to 70-fold with 4 mg of imipenem per kg given at the time of challenge. Nevertheless, protection was quantitatively restored upon concurrent administration of neutralizing anti-TNF-alpha antibody or 4 mg of dexamethasone per kg to these TNF-alpha-hypersensitive mice. Importantly, protection afforded by dexamethasone was not seen when the animals were challenged with viable organisms but without the concurrent administration of antibiotic. An early TNF-alpha response could also be demonstrated upon challenge with Escherichia coli, but in this instance, neither the timing nor the magnitude of that response was influenced by treatment with these antibiotics. We conclude from these studies that the inflammatory response to viable versus killed bacteria may differ markedly depending on the particular bacterium, host sensitivity to TNF-alpha, and possibly the Gram stain classification.

Animals↗

PubMed2000

Differential tumor necrosis factor alpha expression and release from peritoneal mouse macrophages in vitro in response to proliferating gram-positive versus gram-negative bacteria.

Viable Escherichia coli and Staphylococcus aureus bacteria elicited markedly different in vitro tumor necrosis factor alpha (TNF-alpha) responses when placed in coculture with peritoneal murine macrophages. These include quantitative differences in TNF-alpha mRNA expression and corresponding protein product secretion as well as kinetic differences in the profiles of the TNF-alpha responses. Further, lipopolysaccharide (from E. coli) is a major contributing factor to these differences, as revealed by comparative experiments with endotoxin-responsive (C3Heb/FeJ) and endotoxin-hyporesponsive (C3H/HeJ) macrophages. Nevertheless, the eventual overall magnitude of the TNF-alpha secretion of macrophages in response to S. aureus was at least equivalent to that observed with E. coli, while appearing at time periods hours later than the E. coli-elicited TNF-alpha response. Both the magnitude and kinetic profile of the TNF-alpha responses were found to be relatively independent of the rate of bacterial proliferation, at least to the extent that similar results were observed with both viable and paraformaldehyde-killed microbes. Nevertheless, S. aureus treated in culture with the carbapenem antibiotic imipenem manifests markedly altered profiles of TNF-alpha response, with the appearance of an early TNF-alpha peak not seen with viable organisms, a finding strikingly similar to that recently reported by our laboratory from in vivo studies (R. Silverstein, J. G. Wood, Q. Xue, M. Norimatsu, D. L. Horn, and D. C. Morrison, Infect. Immun. 68:2301-2308, 2000). In contrast, imipenem treatment of E. coli-cocultured macrophages does not significantly alter the observed TNF-alpha response either in vitro or in vivo. In conclusion, our data support the concept that the host inflammatory response of cultured mouse macrophages in response to viable gram-positive versus gram-negative microbes exhibits distinctive characteristics and that these distinctions are, under some conditions, altered on subsequent bacterial killing, depending on the mode of killing. Of potential importance, these distinctive in vitro TNF-alpha profiles faithfully reflect circulating levels of TNF-alpha in infected mice. These results suggest that coculture of peritoneal macrophages with viable versus antibiotic-killed bacteria and subsequent assessment of cytokine response (TNF-alpha) may be of value in clarifying, and ultimately controlling, related host inflammatory responses in septic patients.

Animals↗

PubMed2000

Differential expression of caveolin-1 in lipopolysaccharide-activated murine macrophages.

Five reciprocal cycles of subtractive hybridization using cDNA generated from fibroblasts with normal lipopolysaccharide (LPS) responsiveness (lps(n)) and from hyporesponsive (lps(d)) fibroblasts have led to the finding that caveolin-1 is expressed at markedly higher levels of mRNA in lps(d) than in lps(n) fibroblasts. Caveolin-1 message can also be readily detected via reverse transcription-PCR in the RAW264.7 and J774.1 macrophage-like cell lines as well as in primary thioglycolate (TG)-elicited mouse peritoneal macrophages. In RAW264.7 cells, both caveolin-1 mRNA and protein levels are down-regulated by LPS. In TG-elicited C3HeB/FeJ peritoneal macrophages, in contrast, expression of both caveolin-1 protein and mRNA is up-regulated in vitro in response to LPS stimulation. The up-regulation of caveolin-1 protein expression in C3HeB/FeJ peritoneal macrophages can be demonstrated at concentrations as low as 1.0 pg of LPS/ml. However, LPS concentrations approximately 4 orders of magnitude higher (10(4) pg/ml) were required to stimulate the LPS-hyporesponsive C3H/HeJ mice peritoneal macrophages such that significant caveolin-1 protein up-regulation was detected. Caveolin-1, a principal component of plasmalemmal caveolae, has been reported as a potentially important regulator for signal transduction during cellular stimulation. The results described in this report suggest that caveolin-1 expression may be associated with LPS signaling/internalization.

Animals↗

PubMed2000

A comparative trial of imipenem versus ceftazidime in the release of endotoxin and cytokine generation in patients with gram-negative urosepsis. Urosepsis Study Group.

Evidence from in vitro experiments and animal and human studies indicate that antibiotic therapy may induce the release of endotoxin from the outer membrane of Gram-negative bacteria. Antibiotics that bind preferentially to penicillin-binding protein-2 (PBP-2)--such as imipenem--are associated with little release of endotoxin, while antibiotics that preferentially bind to PBP-3--such as ceftazidime--are associated with far greater release of endotoxin. We conducted a randomized, multicenter, double-blind study comparing imipenem to ceftazidime in patients with urinary tract infections caused by Gram-negative bacilli associated with signs and symptoms of systemic inflammation. A total of 33 patients were randomized to receive either imipenem (n = 14) or ceftazidime (n = 19) for initial treatment for urosepsis. No differences in plasma endotoxin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) or urine endotoxin, IL-6 or IL-8 levels were found between the two treatment groups within the first 8 h after antibiotic administration. We conclude that, if differences exist with respect to endotoxin release by these two antimicrobial agents, these differences are not readily demonstrable in this clinical study with carefully defined patients with Gram-negative urinary tract infections.

Adult↗

PubMed1999

Cutting edge: bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages.

LPS is well recognized for its potent capacity to activate mouse macrophages to produce NO, an important inflammatory mediator in innate host defense. We demonstrate here that, although inducing little NO alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce NO in cultures of RAW 264.7 macrophages. The effects of the DNA are mimicked by synthetic CpG-containing oligodeoxynucleotides but not by non-CpG-containing oligodeoxynucleotides. This synergistic activity is not inhibited by neutralizing Abs against IFN. Preincubation of macrophages with DNA for 8-24 h suppresses subsequent synergistic macrophage responses to DNA/LPS, whereas prolonged pretreatment with LPS enhances synergy. RT-PCR analysis indicates that the mRNA levels of the inducible NO synthase gene are also coordinately suppressed or induced. These findings indicate that temporally controlled, synergistic interactions exist between microbial DNA and LPS in the induction of macrophage NO via enhanced inducible NO synthase gene expression.

Animals↗

PubMed1999

Structure-function relationships of bacterial endotoxins. Contribution to microbial sepsis.

A substantial body of knowledge has emerged over the past several decades concerning the primary and tertiary, and quaternary structure of endotoxic LPS and their contribution to the pathogenesis of gram-negative sepsis; however, important questions remain. Among them are the precise three-dimensional configuration of the LPS macromolecule and the contribution of the quaternary structure to the ability of these potent microbial factors to interact with host humoral and cellular inflammatory mediator systems. Also remaining to be sufficiently addressed is the relative contribution of endotoxin interactions with the host to the overall manifestation of disease and conditions under which such contributions serve as the pivotal event in determining outcome. The answers to these questions can be expected to provide valuable insights into potential novel therapeutic intervention strategies and approaches that will ultimately reduce both morbidity and mortality in infection from gram-negative microbes.

Animals↗

PubMed1999

Lipopolyamines: novel antiendotoxin compounds that reduce mortality in experimental sepsis caused by gram-negative bacteria.

The interactions of lipopolyamines, a class of structurally unique compounds currently being used as transfection (lipofection) agents, with lipopolysaccharide (LPS) have been characterized. Our studies have demonstrated that 1,3-di-oleoyloxy-2-(6-carboxyspermyl)-propylamide), available commercially as DOSPER, binds to purified LPS with an affinity of about 1/10 that of polymyxin B. This essentially nontoxic compound inhibits, in a dose-dependent manner, LPS-induced activation of the Limulus clotting cascade and the production of tumor necrosis factor alpha (TNF-alpha) interleukin-6 (IL-6), and nitric oxide from LPS-stimulated J774.A1 cells, a murine macrophage-like cell line. Cytokine inhibition is paralleled by decreased steady-state levels of TNF-alpha and IL-6 mRNA and inhibits the nuclear translocation of nuclear factor kappa B. These findings suggest that the lipopolyamine compound sequesters LPS, thereby blocking downstream cellular activation events that lead to the production of proinflammatory mediators. Administration of DOSPER to D-galactosamine-sensitized mice challenged either with LPS or with Escherichia coli organisms provided significant protection against lethality both with and without antibiotic chemotherapy. Partial protection is evident in LPS-challenged mice treated with DOSPER as late as 2 to 4 h following the endotoxin challenge. A greater degree of protection is observed in E. coli-challenged animals receiving ceftazidime than in those receiving imipenem, which is probably attributable to the higher levels of LPS released in vivo by the former antibiotic. Potent antiendotoxic activity, low toxicity, and ease of synthesis render the lipopolyamines candidate endotoxin-sequestering agents of potential significant therapeutic value.

Animals↗

PubMed1998

Differential regulation of lipopolysaccharide (LPS) activation pathways in mouse macrophages by LPS-binding proteins.

LPS binding to its receptor(s) on macrophages induces the synthesis of inflammatory mediators involved in septic shock. While the signaling mechanism(s) remains to be fully defined, the human LPS-binding protein (LBP) is known to regulate responses to LPS by facilitating its binding to CD14 on human monocytes. The structurally related bactericidal permeability increasing protein (BPI) differs from LBP by inhibiting LPS-induced human monocyte activation. We have demonstrated that, unlike the human monocyte response to LPS, both LBP and BPI inhibited LPS-stimulated TNF-alpha production in mouse peritoneal macrophages. In contrast, LPS-dependent nitric oxide release was not affected by LBP. LPS induces the phosphorylation of a number of proteins in a dose and time-dependent manner, however, the pattern of LPS-induced phosphorylation was not reduced by either LBP or BPI under conditions that result in selective TNF-alpha inhibition. Further, activation of the transcription factor NF-kappaB in response to LPS was also not modified by either LBP or BPI. Finally, no differences were detected in TNF-alpha or inducible nitric oxide synthase mRNA accumulations induced by LPS in the presence or absence of either protein, whereas a slight decreased mRNA stability was observed in the group with LPS treatment. These results would suggest that many of the early signaling events contribute to LPS-induced macrophage signaling at a point preceding the divergence of pathways that differentially regulate TNF-alpha and NO production.

Acute-Phase Proteins↗

PubMed1998

Reprogramming of lipopolysaccharide-primed macrophages is controlled by a counterbalanced production of IL-10 and IL-12.

We studied the potential role of a cytokine regulatory mechanism(s) in LPS-dependent reprogramming and modulation of TNF-alpha and nitric oxide (NO) responses in mouse peritoneal macrophages. Reciprocal regulation of TNF-alpha and NO production by LPS-primed and LPS-stimulated macrophages was found to be dependent on the presence of soluble secretory products released by the cells during the initial LPS priming interaction. Pretreatment of naive macrophages with different mouse recombinant cytokines such as rIL-10, rIL-12, and rIFN-gamma dose dependently and differentially regulated subsequent LPS-induced production of TNF-alpha, IL-6, and NO by cytokine-primed cells. Analysis of IL-12 and IL-10 levels present in culture supernatants of LPS-primed and LPS-stimulated macrophages revealed a high degree of correlation between the profiles of TNF-alpha and IL-12 as well as NO and IL-10. Furthermore, LPS priming of macrophages in the presence of anti-IL-12-neutralizing mAb attenuated TNF-alpha responses while at the same time up-regulated NO production. In contrast, neutralization of endogenous IL-10 with anti-IL-10 mAb resulted in considerable TNF-alpha response at LPS priming doses under conditions that would otherwise strongly inhibit TNF-alpha production. We also found that the initial LPS priming of naive macrophages differentially and dose dependently regulates expression of mRNAs for IL-10, IL-12, and IFN-gamma in LPS-primed macrophages. Collectively, our data provide experimental support for the hypothesis that a cytokine regulatory network, most probably autocrine, tightly controls the reciprocal modulation of TNF-alpha and NO responses in LPS-primed macrophages.

Animals↗

PubMed1998

Correlation of antibiotic-induced endotoxin release and cytokine production in Escherichia coli-inoculated mouse whole blood ex vivo.

Escherichia coli were incubated in mouse whole blood ex vivo supplemented with beta-lactam antibiotics that possessed preferential affinities for penicillin-binding proteins (PBPs). After 4 h, viable bacteria were undetectable in the presence of any of the 3 antibiotics tested, whereas significant increases in colony-forming units were detected in samples not treated with antibiotics. Differential levels of endotoxin in platelet-rich plasma were detected using the limulus amebocyte lysate assay, according to differential antibiotic affinities for the various PBPs. Levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in antibiotic-treated cultures after 8 h of incubation correlated well with the levels of endotoxin at 4 h (r = .96, P < .0001 for TNF-alpha; r = .91, P = .0002 for IL-6). These data indicate that differential affinities of beta-lactam antibiotics for PBPs affect both endotoxin and cytokine responses ex vivo in mouse blood and correlate with in vivo protective efficacy of these antibiotics in gram-negative experimental models.

Animals↗