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.

Mechanisms involved in the pathogenesis of sepsis are not necessarily reflected by in vitro cell activation studies.

It is thought that lipopolysaccharide (LPS) from gram-negative bacteria contributes significantly to the pathogenesis of septic shock. In vitro studies to address the mechanisms involved in this process have often investigated human monocytes or mouse macrophages, since these cells produce many of the mediators found in septic patients. Targeting of these mediators, especially tumor necrosis factor alpha (TNF-alpha), has been pursued as a means of reducing mortality in sepsis. Two experimental approaches were designed to test the assumption that in vitro studies with macrophages accurately predict in vivo mechanisms of LPS pathogenesis. In the first approach, advantage was taken of the fact that on consecutive days after injection of thioglycolate into mice, increased numbers of macrophages could be harvested from the peritoneum. These cells manifested markedly enhanced levels of in vitro TNF-alpha, interleukin 6 (IL-6), and nitric oxide production in response to LPS. In D-galactosamine-sensitized mice, however, thioglycolate treatment significantly decreased mortality due to LPS, as well as levels of circulating TNF-alpha and IL-6. Anti-TNF-alpha treatment confirmed this cytokine's role in the observed lethality. In a second experimental approach, we compared the mouse macrophage-stimulating potencies of different LPS preparations with their lethalities to mice. In these studies, the in vitro macrophage-stimulating profiles presented by rough-LPS and smooth-LPS preparations were the reverse of their relative lethal potencies in vivo. In conclusion, peritoneal macrophages appear not to be the major cells responsible for the overall host response during endotoxic shock. These findings underscore the importance of verifying the correlation of in vivo systems with in vitro systems when attributing specific functions to a cell type.

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.

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.

Two functionally independent pathways for lipopolysaccharide-dependent activation of mouse peritoneal macrophages.

We have investigated the effects of human LPS-binding protein (LBP) and human bactericidal/permeability-increasing protein (BPI) on LPS-dependent activation of mouse thioglycolate-elicited peritoneal macrophages in vitro, in comparison with human PBMCs. Confirming earlier published studies, BPI inhibited, and LBP enhanced, the ability of LPS to stimulate PBMC production of the cytokines TNF-alpha and IL-6. In marked contrast to these results, under identical conditions of in vitro culture, both LBP and BPI suppressed, in a dose-dependent manner, the ability of LPS to stimulate cytokine production in mouse macrophages. Further, while human BPI also suppressed LPS-dependent NO secretion in mouse macrophages, human LBP had no inhibitory effect on NO secretion under conditions that inhibited TNF-alpha secretion. These data provide the first direct evidence that mouse macrophages may utilize two independent pathways in response to LPS, thus leading to different phenotypic responses.

Tumor necrosis factor alpha plus interleukin 1 beta treatment protects granulocytopenic mice from Pseudomonas aeruginosa lung infection: role of an unusual inflammatory response.

We have recently demonstrated that treatment with interleukin 1 beta (IL-1 beta) plus tumor necrosis factor alpha (TNF alpha) protects granulocytopenic hosts from Pseudomonas aeruginosa aerosol challenge. In this study we characterized the inflammatory response induced by P. aerugionsa in granulocytopenic mice treated with 2,000 U IL-1 beta plus 2,000 U TNF alpha. Treatment with the nonsteroidal anti-inflammatory agent piroxicam abolished both the protective effect of cytokine treatment and the increase in myeloperoxidase (MPO) pulmonary activity. Histopathological studies revealed that, after aerosol challenge with P. aeruginosa, treatment with these cytokines induced migration and extravasation of mononuclear cells of immature appearance into the lung parenchyma. These cells contained MPO in their cytoplasm and displayed phagocytic capacity. Resident alveolar macrophages exhibited signs of activation and appeared in reduced numbers in bronchoalveolar lavage fluid. We suggest that the inflammatory response promoted by low TNF alpha plus IL-1 beta doses may be one mechanism responsible for protection of granulocytopenic hosts from P. aeruginosa aerosol challenge.

Modulation of human polymorphonuclear leukocyte chemotaxis and superoxide anion production by Pseudomonas aeruginosa exoproducts, IL-1 beta and piroxicam.

Whereas addition of 200 ng ml-1 exotoxin A (exoA) did not modify PMNL chemotaxis, 20 U ml-1 human recombinant interleukin-1 beta (hrIL-1 beta) primed polymorphonuclear leukocytes (PMNL) for migration towards Pseudomonas aeruginosa peptide chemotactins (PAPCs). Piroxicam (100 micrograms ml-1), a non-steroidal anti-inflammatory agent (NSAIA), inhibited PMNL chemotaxis and abolished the priming effect of hrIL-1 beta. Both PAPCs and exoA induced PMNL superoxide anion production, but neither hrIL-1 beta nor piroxicam modified significantly PMNL superoxide anion production induced by PAPCs. The fact that hrIL-1 beta can prime PMNL for chemotaxis towards PAPCs and that piroxicam can abolish activation by primed PMNL are findings relevant to the pharmacological control of lung tissue damage during P. aeruginosa pneumonia.

The effect of treatment with interleukin-1 and tumor necrosis factor on Pseudomonas aeruginosa lung infection in a granulocytopenic mouse model.

The efficacy of treatment with interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) on Pseudomonas aeruginosa pneumonia was evaluated in a granulocytopenic mouse model. Combined intravenous administration of 2000 U IL-1 beta plus 2000 U TNF alpha significantly diminished mortality from aerosol challenge with P. aeruginosa. Mice treated with IL-1 beta, TNF alpha, or both also exhibited a significant enhancement in pulmonary clearance of P. aeruginosa. Combined cytokine administration induced an increase in the pulmonary content of myeloperoxidase activity. Mature leukocytes were not detected in either circulation or bronchoalveolar lavage fluid from granulocytopenic, cytokine-treated mice. In conclusion, IL-1 beta and TNF alpha treatment exhibited a synergistic protective effect from pulmonary P. aeruginosa challenge in granulocytopenic hosts, probably due to enhancement of nonspecific antibacterial mechanisms.

The effect of a cyclooxygenase inhibitor on the in vivo polymorphonuclear leukocyte migration to Pseudomonas aeruginosa peptide chemotactins.

Pseudomonas aeruginosa chemotactic peptides (PAPCs) induced migration of polymorphonuclear leukocytes (PMNL) into the lungs when administered by the aerosol route. Migration of PMNL into the lungs and total protein content of lung lavage fluids in response to PAPCs aerosol challenge, and mortality from lethal challenge with P. aeruginosa were decreased by piroxicam treatment.

Piroxicam-copper complexes: inhibition of polymorphonuclear leukocyte migration to Pseudomonas aeruginosa chemotactins in vivo and superoxide dismutase-like activity in vitro.

Piroxicam-copper (Cu2+) complexes, formed spontaneously by mixing solutions of piroxicam and CuSO4 (1:1 Cu2+:piroxicam), inhibited the superoxide anion-catalyzed reduction of ferricytochrome C in a dose-related fashion. Addition of ethylenediaminetetraacetate to the mixture decreased in a dose-related manner the superoxide dismutase (SOD)-like activity of piroxicam-Cu2+. Piroxicam alone (10(-5) M, final concentration) did not display SOD-like activity but 10(-5) M Cu2+ exhibited significant activity, similar to that of piroxicam-Cu2+. Intraperitoneal treatment of mice with either 0.64 mg/kg piroxicam or its Cu2+ complexes (0.64 mg/kg piroxicam + 0.12 mg/kg Cu2+) was equally effective in diminishing both the migration of polymorphonuclear leukocytes (PMNL) to the airways and the content of myeloperoxidase activity in the lungs, induced by aerosol challenge with Pseudomonas aeruginosa peptide chemotactins. Therefore, piroxicam-Cu2+ complexes may provide both the anti-inflammatory activity of piroxicam plus the SOD-like activity of Cu2+.

Treatment with a non-steroidal anti-inflammatory agent delays the growth of spontaneous pulmonary metastases of a mammary adenocarcinoma of non-detected immunogenicity.

Previous reports showed that treatment with non-steroidal anti-inflammatory agents (NSAIA) can alter the growth profile of a variety of tumours. In this study, the effect of NSAIA treatment on the growth of the primary tumour and the appearance of spontaneous pulmonary metastases, was investigated. A mammary adenocarcinoma of non-detected immunogenicity, C7HI, was grafted subcutaneously in the lateral flank of Balb/c mice. Oral treatment with approximately 1 mg kg-1 day-1 piroxicam delayed both tumour growth and the growth of pulmonary metastases. Survival of mice bearing the primary tumour was significantly lengthened by anti-inflammatory treatment. Similarly, in separate experiments, after surgical removal of the primary tumour by day 34 after grafting, the group of mice treated orally with piroxicam also exhibited a higher survival rate than the control group. Upon surgical removal of the primary tumour 34 days after grafting, piroxicam treatment significantly decreased both the number and size of pulmonary metastases. The results of this study lends support to the hypothesis that inhibition or modulation of inflammation may delay tumour organisation and growth. It is suggested that piroxicam treatment may be an appropriate adjunct therapy to delay the appearance of pulmonary metastases and to increase life-expectancy in a host whose primary tumour has to be surgically removed.

Preliminary characterization of Pseudomonas aeruginosa peptide chemotactins for polymorphonuclear leukocytes.

In a previous report, we showed that supernatants of Pseudomonas aeruginosa cultures exhibit chemotactic activity for polymorphonuclear leukocytes (PMNL). In this study, P. aeruginosa chemotactins were isolated, purified, and partially characterized. The organisms were cultured in Vogel-Bonner defined medium, and cultures were stopped in late log phase. Chemotactins withstood heating, remained unaltered after acid or alkali treatment in a pH range from 4 to 10, and resisted digestion by trypsin or carboxypeptidase, but chemotactic activity was decreased by 73% after incubation with pronase. Only 2% of the total chemotactic activity of culture supernatants could be extracted with chloroform. Chemotactins with molecular sizes less than 3 kDa constituted the largest contribution to the chemotactic activity of culture supernatants. Pretreatment of PMNL with 10(-5) M formylmethionyl-leucyl-phenylalanine (FMLP) inhibited chemotaxis towards FMLP and P. aeruginosa culture supernatants but not towards complement component C5a. In conclusion, the total chemotactic activity for PMNL of P. aeruginosa culture supernatants was due, almost exclusively, to chemotactins that have properties similar, if not identical, to those exhibited by formylmethionyl peptides.