Biomedical subjects
G Warr
Publications and source records attributed to G Warr.
LPS down-regulates the expression of chemokine receptor CCR2 in mice and abolishes macrophage infiltration in acute inflammation.
Interactions between chemokines and their specific receptors are important for leukocyte trafficking. The CC-chemokine monocyte chemoattractant protein-1 (MCP-1) and its specific receptor CCR2 are essential in monocytic infiltration and have been associated with several inflammatory diseases. It has been reported that several endotoxin and proinflammatory cytokines inhibit CCR2 expression in vitro in human monocytes. We report here that lipopolysaccharides (LPS) down-regulated CCR2 expression both in vitro and in vivo. Injection of LPS into mice dramatically reduced the expression of CCR2 on the surface of peripheral blood cells and completely blocked macrophage infiltration into the peritoneal cavity in response to thioglycollate elicitation. In addition, treatment of mice with LPS reduced their efficiency to clear Listeria monocytogenes infection. These results suggest that down-regulation of CCR2 and blockage of monocyte infiltration may contribute to the inhibition of macrophage function in vivo by a low dose of LPS.
Influence of the mu-chain C-terminal sequence on polymerization of immunoglobulin M.
Immunoglobulin (IgM) is found in various states of covalent polymerization (microL)n, where n is typically 8, 10, or 12. The usual form of IgM of bony fish is tetrameric (8 microL units) as compared to the pentameric form (10 microL units) observed in cartilaginous fish and mammals. Two hypotheses were tested in this study. First, that the length of the mu-chain C terminus following Cys575 determines whether an IgM polymerizes as a tetramer or as a pentamer. This was tested by examining the covalent polymerization state of mouse IgM mutated to contain a series of mu-chain C-termini from bony and cartilaginous fish. The results proved this hypothesis wrong: mouse IgM bearing the C-terminal sequence of shark, salmon and cod mu-chain behaved identically to native mouse IgM, forming predominantly (microL)10 and (microL)12 forms. The second hypothesis was that an additional Cys residue near the C terminus of the mu-chain is responsible for the multiple covalent structures seen in IgM of the channel catfish. The addition of a catfish C terminus to the mouse mu-chain resulted, as predicted, in the production of a series of covalently bonded forms, with the major species being (microL)4. When a Ser-Cys unit was removed from the catfish C terminus added to the mouse mu-chain, this resulted in production of IgM indistinguishable in structure from that of wild-type mouse IgM.
Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor.
The CC-chemokine receptor CCR5 has been shown to be the major coreceptor for HIV-1 entry into cells, and humans with homozygous mutation in the ccr5 gene are highly resistant to HIV-1 infection, despite the existence of many other HIV-1 coreceptors. To investigate the physiologic function of CCR5 and to understand the cellular mechanisms of these clinical observations, we generated a CCR5-deficient mouse model (ccr5[-/-]) by targeted deletion of the ccr5 gene. We found that although developed normally in a pathogen-free environment, CCR5-deficient mice showed reduced efficiency in clearance of Listeria infection and exert a protective effect against LPS-induced endotoxemia, reflecting a partial defect in macrophage function. In addition, CCR5-deficient mice had an enhanced delayed-type hypersensitivity reaction and increased humoral responses to T cell-dependent antigenic challenge, indicating a novel role of CCR5 in down-modulating T cell-dependent immune response.
Multiple hemopoietic defects and lymphoid hyperplasia in mice lacking the transcriptional activation domain of the c-Rel protein.
The c-rel protooncogene encodes a member of the Rel/nuclear factor (NF)-kappaB family of transcriptional factors. To assess the role of the transcriptional activation domain of c-Rel in vivo, we generated mice expressing a truncated c-Rel (Deltac-Rel) that lacks the COOH-terminal region, but retains a functional Rel homology domain. Mice with an homozygous mutation in the c-rel region encoding the COOH terminus of c-Rel (c-relDeltaCT/DeltaCT) display marked defects in proliferative and immune functions. c-relDeltaCT/DeltaCT animals present histopathological alterations of hemopoietic tissues, such as an enlarged spleen due to lymphoid hyperplasia, extramedullary hematopoiesis, and bone marrow hypoplasia. In older c-relDeltaCT/DeltaCT mice, lymphoid hyperplasia was also detected in lymph nodes, liver, lung, and stomach. These animals present a more severe phenotype than mice lacking the entire c-Rel protein. Thus, in c-relDeltaCT/DeltaCT mice, the lack of c-Rel activity is less efficiently compensated by other NF-kappaB proteins.
Functional and molecular characterization of teleost leukocytes.
The coupling of immunologically relevant in vitro assay systems, cell separation techniques, and the development of distinct clonal leukocyte lines has established the existence of T, B, natural killer, and accessory cell equivalents in teleosts. B cells are directly defined by monoclonal antibodies to teleost immunoglobulin (Ig) and identification of Ig H and L chain genes. As in mammals, fish B cells show Ig H-chain gene rearrangements, allelic exclusion, produce both membrane-bound and secreted forms of Ig, and transduce intracellular proliferative signals upon anti-Ig cross-linking. It has also been found that some fish B cells express a unique chimeric Ig chain with sequence homology to mammalian Ig delta. Teleost T cells are still indirectly defined as sIg- lymphocytes due to a lack of definitive surface markers. These mIg- lymphocytes are the responding cells in mixed leukocyte cultures, proliferate specifically to autologously processed and presented antigen, provide helper function for in vitro antibody responses, and produce interleukin-like factors upon activation. Recent identification of teleost T-cell receptor alpha and beta genes has now permitted the unequivocal genetic demonstration that some of these mIg- cells are bona fide T cells. It is anticipated that such long-term clonal cell lines will be indispensable tools for dissecting the physiology, biochemistry and molecular biology of teleost immune responses.
Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor.
Chemokines are a structurally related family of cytokines that are important for leukocyte trafficking. The C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is a potent monocyte activator in vitro and has been associated with monocytic infiltration in several inflammatory diseases. One C-C chemokine receptor, CCR2, has been identified that mediates in vitro responses to MCP-1 and its close structural homologues. CCR2 has also recently been demonstrated to be a fusion cofactor for several HIV isolates. To investigate the normal physiological function of CCR2, we generated mice with a targeted disruption of the ccr2 gene. Mice deficient for CCR2 developed normally and had no hematopoietic abnormalities. However, ccr2(-/-) mice failed to recruit macrophages in an experimental peritoneal inflammation model. In addition, these mice were unable to clear infection by the intracellular bacteria, Listeria monocytogenes. These results suggest that CCR2 has a nonredundant role as a major mediator of macrophage recruitment and host defense against bacterial pathogens and that MCP-1 and other CCR2 ligands are effectors of those functions.
Multifocal defects in immune responses in RelB-deficient mice.
Mice with a targeted disruption of the Rel/nuclear factor-kappaB family member RelB develop a complex inflammatory phenotype, myeloid hyperplasia, and splenomegaly due to extramedullary hemopoiesis. In this work, we report that RelB-deficient mice, in addition to the pathologic changes, were highly susceptible to infection by the facultative intracellular bacterium Listeria monocytogenes. RelB binds transcriptionally active kappaB motifs in the TNF-alpha promoter in normal cells, and in vitro studies with macrophages isolated from RelB-deficient animals revealed impaired production of TNF-alpha in response to LPS and IFN-gamma. RelB-deficient mice also were unable to mount a protective immune response against lymphocytic choriomeningitis virus. These results indicate a defective T cell-macrophage interaction and cytotoxic T cell response, respectively, in mice lacking RelB. Analysis of resting and specific Ab production demonstrated that while RelB is not required for the secretion of Ig isotypes that result from heavy chain class switching, it is necessary for normal production of Ag-specific IgG in response to T cell-dependent and -independent stimuli. Thus, RelB is not only essential for a normal hemopoietic system in the unchallenged animal, but also involved in various specific and nonspecific immune responses.
Trichodimerol (BMS-182123) inhibits lipopolysaccharide-induced eicosanoid secretion in THP-1 human monocytic cells.
The fungal metabolite trichodimerol (BMS-182123) has demonstrated inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha) secretion in various in vitro macrophage models (human and murine) including primary and tumor cell lines. When challenged with LPS, differentiated THP-1 monocytic cells secrete elevated levels of the cyclooxygenase products prostaglandin E2 (PGE2), thromboxane B2, and prostaglandin F2alpha (PGF2alpha). Studies directed at elucidating the mechanism of action of BMS-182123 as a TNF-alpha inhibitor revealed that the compound has a profound inhibitory effect on prostanoid secretion in response to LPS challenge. The key enzymes in prostaglandin synthesis are the constitutive cyclooxygenase, prostaglandin H synthase-1 (PGHS-1), and the mitogen-induced cyclooxygenase (PGHS-2), which is induced upon LPS stimulation in THP-1 cells. BMS-182123 did not inhibit the cyclooxygenase activity of PGHS-1 in an in vitro assay, suggesting that inhibition is due to a blockade in synthesis of cyclooxygenase enzyme. Western blot analysis of microsomal pellets from THP-1 cells stimulated with LPS (with or without BMS-182123 pretreatment) provided convincing evidence that the inhibition of prostaglandin synthesis is a result of suppressed synthesis of PGHS-2 enzyme. Northern blot analysis of THP-1 RNA demonstrated that BMS-182123 inhibits the induction of PGHS-2 at the level of transcription.
In vitro and in vivo antibacterial activities of BMY 40062, a new fluoronaphthyridone.
The in vitro and in vivo activities of a new naphthyridone, BMY 40062, were compared with those of ciprofloxacin and ofloxacin. BMY 40062 showed about threefold more activity than ciprofloxacin showed and four- to eightfold more activity than ofloxacin showed against staphylococci, streptococci, and enterococci. BMY 40062 showed generally twofold less activity than ciprofloxacin showed against most species of the family Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. but twofold more activity than ofloxacin showed against these organisms. BMY 40062 and ofloxacin were more active than ciprofloxacin against Bacteroides fragilis and Clostridium difficile. The antiureaplasmal and antichlamydial activities of BMY 40062 were similar to those of the tetracyclines and were 4- and 16-fold, respectively, higher than those of ciprofloxacin. The in vitro activities of BMY 40062 were influenced by pH and magnesium, although these factors appeared to affect the activity of BMY 40062 against P. aeruginosa to a lesser extent than those of ciprofloxacin and ofloxacin. BMY 40062 was found to be bactericidal, and cross-resistance with other fluoroquinolones was observed. In mouse protection tests, the efficacy of BMY 40062 reflected its in vitro potency. BMY 40062 exhibited longer half-life, higher maximum concentration in serum, greater area under the curve, and better bioavailability in mice after oral dosing than ciprofloxacin. Compared with ofloxacin, BMY 40062 had a lower maximum concentration in serum but a much longer half-life in mice. BMY 40062 was more effective than ciprofloxacin and ofloxacin in penetrating mouse macrophages and killing macrophage-associated Staphylococcus aureus.
Induction of tumor necrosis factor, IFN-gamma, and acute lethality in mice by toxic and non-toxic forms of lipid A.
The effect of the i.v. administration of endotoxin (LPS), diphosphoryl lipid A, and the non-toxic derivative monophosphoryl lipid A (MPL), on the production of serum cachectin (TNF), IFN-gamma, and the appearance of endotoxin shock have been measured in mice primed with Listeria monocytogenes. All three of the lipid A varieties induced the production of TNF in a dose-dependent manner. Although comparable peak levels of TNF were produced (5 X 10(5) to 1 X 10(6) U/ml), treatments with LPS or diphosphoryl lipid A were lethal whereas those with MPL were not. A study following TNF in the mouse sera for up to 24 h after treatment with the lipid A types showed that serum levels of TNF peaked 90 min after the treatment, and that TNF levels induced by LPS treatment were maintained for several hours longer than those induced by lipid A or MPL. All three molecules also resulted in the production of IFN-gamma in the serum, which peaked at 4 to 5 h after treatment. After 90 min there were no significant differences in the levels of serum IFN-gamma in any of the groups of treated animals. However, as was observed with the TNF, the levels of IFN in animals treated with LPS persisted longer than those induced by MPL or lipid A. These results suggest that the non-toxic MPL as well as the toxic forms of lipid A can induce the production of TNF by macrophages. Furthermore, although it is essential, TNF alone is not necessarily sufficient to induce septic shock in mice.
Antigenically important regions of the relaxin molecule: studies with monoclonal antibodies.
A panel of 22 monoclonal antibodies was produced against porcine relaxin. Antibodies were shown to be specific for porcine relaxin and appeared to recognize antigenic determinants associated with at least two different areas of the molecular surface, one of which was the N-terminal region of the A chain.
Further studies on the effect of a transplanted methylcholanthrene-induced fibrosarcoma on humoral immunity in CBA mice.
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Induction of aryl hydrocarbon hydroxylase in human lymphocytes and pulmonary alveolar macrophages--a comparison.
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Chemotaxis of human leukocytes: responsiveness to Mycoplasma pneumoniae.
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