Analysis of cytokine immune response profile in response to inflammatory stimuli in mice with genetic defects in fetal and adult hemoglobin chain expression.

Injections of a crude fetal sheep liver extract (FSLE) containing fetal hemoglobin, MPLA, and glutathione (GSSH) reversed cytokine changes in aged mice. To investigate the role of fetal hemoglobin we derived mice with homzygous deletions for either of the two major ßchains, HgbßmaKO or HgbßmiKO. Hgbßmi is the most prominent fetal Hgbß chain, with Hgbßma more prominent in adult mice. Mice lacking another fetal Hgb chain, HgbεKO, died in utero. CHO cells transfected with cloned Hgb chains were used to produce proteins for preparation of rabbit heteroantibodes. Splenocytes from HgbßmaKO mice stimulated in vitro with Conconavalin A showed a higher IL-2:IL-4 ratio than cells from HgbßmiKO mice. Following immunization in vivo with ovalbumin in alum, HgbßmaKO mice produced less IgE than HgbßmiKO mice, suggesting that in the absence of HgbßmiKO mice had a predeliction to heightened allergic-type responses. Using CHO cells transfected with cloned Hgb chains, we found that only the fetal Hgb chain, Hgbε, was secreted at high levels. Secretion of Hgbßma or Hgbßmi chains was seen only after genetic mutation to introduce the two N-linked glycosylation sites present in Hgbε, but absent in the Hgbß chains. We speculated that a previously unanticipated biological function of a naturally secreted fetal Hgb chain may be partly responsible for the effects reported following injection of animals with fetal, not adult, Hgb. Mice receiving injections of rabbit anti-Hgbε but not either anti-Hgbßma or anti-Hgbßmi from day 14 gestation also showed a bias towards the higher IL-2:IL-4 ratios seen in HgbßmiKO mice.

Hyperlipoproteinemic low-density lipoprotein receptor-deficient mice are more susceptible to sepsis than corresponding wild-type mice.

High circulating concentrations of lipoproteins have been shown to modify the cytokine response and reduce mortality after endotoxin or live bacterial challenge. Sepsis, however, is more complex than endotoxemia, and it is not clear whether elevated plasma lipoproteins will be protective. Previous studies have shown that the low-density-lipoprotein receptor deficient (LDLR-/-) mice with increased circulating LDL are protected against the lethal effects of endotoxemia and Gram-negative infection. We evaluated whether the LDLR-/- mice would be protected against the effects of sepsis induced by cecal ligation and puncture (CLP). Mortality was greater in LDLR-/-mice than in control C57Bl/6J mice. At 120 h after inducing sepsis, 20% of the control mice survived whereas none of theLDLR-/-mice were alive. Prior to inducing sepsis, serum concentrations of amyloid A protein and lipopolysaccharide binding protein (LBP) were significantly elevated in the LDLR-/-mice in comparison to the C57Bl/6J mice. Protein expression of sCD14 was also greater in the serum from the LDLR-/-mice than the C57Bl/6J mice. The elevated serum concentrations of LBP and CD14 were not associated with increases in the levels of liver CD14 mRNA and LBP mRNA. After inducing sepsis, serum concentration of interleukin (IL)-1beta was also significantly higher in LDLR-/-mice than in the control C57Bl/6J mice. These findings indicate that the LDLR-/-mice were more susceptible to the lethal effects of sepsis induced by CLP. The LDLR-/-mice also had higher serum concentrations of baseline, acute phase response proteins, SAA and LBP, and increased production of IL-1beta in response to CLP.

The dual role of LBP and CD14 in response to Gram-negative bacteria or Gram-negative compounds.

Innate immunity initiates protection of the host organism against invasion of micro-organisms by specific recognition mechanisms. This article reviews the dual role of LBP/CD14 in innate immunity, focusing mostly on experiments performed in mice by the authors. LPS induces uncontrolled pro-inflammatory response that kills the host and is LBP- and CD14-dependent, as neutralization of LBP and CD14 prevents lethal shock. However, surprisingly, the synthetic Pam3CysSerLys4 bacterial lipoprotein from Escherichia coli (BLP), which is well tolerated in mice, kills the mice upon LBP or CD14 blockade. Furthermore, after blockade of LBP and CD14, the mice succumb to a challenge with virulent Klebsiella pneumoniae or Salmonella typhimurium. Therefore, host responses to Gram-negative bacteria are not identical to that of LPS or BLP. When the host is in the presence of virulent Gram-negative bacteria, the invading pathogens must be held in check by the innate immune system until a specific immune response is mounted. Under these conditions, LBP, CD14, and likely Toll-like receptors (TLRs) are a prerequisite to trigger a pro-inflammatory response of macrophages, which is crucial for keeping an infection under control. These studies indicate that we are very far from understanding how the innate system works and more work needs to be done concerning LBP, CD14 or TLRs. Therefore, caution should be the rule about the use of therapeutic approaches to block the pro-inflammatory response in Gram-negative infections.

Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues.

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L(1) to S(37)) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K(15) to V(32)) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14(+) murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14(+) cells and attenuating production of tumor necrosis factor alpha (TNF-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.

Initial responses to endotoxins and Gram-negative bacteria.

The innate immune system initiates host defence against invasive microbial pathogens using specific recognition mechanisms. Here we review the current concepts and the molecular basis of innate immune responses to bacterial infections, focusing our attention on the actors involved in the response to Gram-negative bacteria. Lipopolysaccharide (LPS) is the major virulence factor of Gram-negative bacteria. During the past decade, enormous progress has been obtained in the elucidation of LPS recognition and signalling in mammalian phagocytes. According to the current model, recognition of LPS is initialized by the cooperative interplay between the LPS-binding protein (LBP), the membrane-bound or soluble forms of CD14 and the recently identified Toll-like receptor 4 (TLR4)-MD-2 complex. Recognition of LPS leads to the rapid activation of an intracellular signalling pathway, highly homologous to the signalling pathway of interleukin-1, which results in the release of pro-inflammatory mediators. In vivo models in which animals are challenged with LPS or Gram-negative bacteria have highlighted opposite roles for LBP, CD14 and TLRs. Regarding LPS challenge, there is a large body of evidence in favour of a detrimental role played by LBP, CD14 and TLRs. These molecules sensitize the host to a LPS-induced uncontrolled acute inflammatory response that results in animal death. However, when the host is in the presence of virulent Gram-negative bacteria, the invading pathogens must be held in check by the innate immune system until a specific immune response is mounted. Under these conditions, LBP, CD14 and TLRs are required to trigger a pro-inflammatory response which is crucial for keeping infection under control. Therefore, caution should be the rule about the development of therapeutic approaches aimed at blocking the pro-inflammatory response during Gram-negative infections.

The role of lipopolysaccharide binding protein in resistance to Salmonella infections in mice.

Polymorphonuclear leukocytes (PMN) and LPS-binding protein (LBP) are both components of the innate immune system. LBP is a plasma protein that binds to lipid A and enhances the biological activity of LPS 100- to 1000-fold. Recently it was reported that LBP-deficient mice are more susceptible to Salmonella typhimurium infection. Here we report that LBP KO mice are more susceptible to Salmonella peritonitis, but not to oral or i.v. infection. LBP knockout (KO) mice responded normally to i.p. injections of Staphylococcus aureus and casein, but not to i.p. injection of S. typhimurium or Salmonella LPS. Mice with a mutation in Toll-like receptor 4 (C3H/HeJ) have a similar defect in PMN chemotaxis. In normal mice S. typhimurium stimulated production of the CXC chemokines macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant, but levels of cytokine-induced neutrophil chemoattractant and macrophage inflammatory protein-2 were greatly reduced in the LBP KO mice. LBP KO mice pretreated with casein to attract PMN in an LBP-independent manner were more resistant to Salmonella infection, but neutropenic mice were not protected by casein. Splenic TNF-alpha mRNA levels were also lower in LBP KO than in control mice infected with Salmonella. Since TNF-alpha can activate PMN, LBP KO mice may have both fewer and less active PMN in the first few hours after Salmonella are injected, making LBP KO mice more susceptible. This work confirms the importance of PMN in resistance to Salmonella infections and shows that this is facilitated by LBP.