Rad51 expression and localization in B cells carrying out class switch recombination.

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells.

In many diseases, tissue hypoxia occurs in conjunction with other inflammatory processes. Since previous studies have demonstrated a role for leukocytes in ischemia/reperfusion injury, we hypothesized that endothelial hypoxia may "superinduce" expression of an important leukocyte adhesion molecule, E-selectin (ELAM-1, CD62E). Bovine aortic endothelial monolayers were exposed to hypoxia in the presence or absence of tumor-necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS). Cell surface E-selectin was quantitated by whole cell ELISA or by immunoprecipitation using polyclonal anti-E-selectin sera. Endothelial mRNA levels were assessed using ribonuclease protection assays. Hypoxia alone did not induce endothelial E-selectin expression. However, enhanced induction of E-selectin was observed with the combination of hypoxia and TNF-alpha (270% increase over normoxia and TNF-alpha) or hypoxia and LPS (190% increase over normoxia and LPS). These studies revealed that a mechanism for such enhancement may be hypoxia-elicited decrements in endothelial intracellular levels of cAMP (<50% compared with normoxia). Addition of forskolin and isobutyl-methyl-xanthine during hypoxia resulted in reversal of cAMP decreases and a loss of enhanced E-selectin surface expression with the combination of TNF-alpha and hypoxia. We conclude that endothelial hypoxia may provide a novel signal for superinduction of E-selectin during states of inflammation.

Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells.

Mammals continually confront microbes at mucosal surfaces. A current model suggests that epithelial cells contribute to defense at these sites, in part through the production of broad-spectrum antibiotic peptides. Previous studies have shown that invertebrates can mount a host defense response characterized by the induction in epithelia] cells of a variety of antibiotic proteins and peptides when they are challenged with microorganisms, bacterial cell wall/membrane components, or traumatic injury [Boman, H.G. & Hultmark, D. (1987) Annu. Rev. Microbiol. 41, 103-126J. However, factors that govern the expression of similar defense molecules in mammalian epithelial cells are poorly understood. Here, a 13-fold induction of the endogenous gene encoding tracheal antimicrobial peptide was found to characterize a host response of tracheal epithelia] cells (TECs) exposed to bacterial lipopolysaccharide (LPS). Northern blot data indicated that TECs express CD14, a well-characterized LPS-binding protein known to mediate many LPS responses. A monoclonal antibody to CD14 blocked the observed tracheal antimicrobial peptide induction by LPS under serum-free conditions. Together the data support that CD14 of epithelial cell origin mediates the LPS induction of an antibiotic peptide gene in TECs, providing evidence for the active participation of epithelial cells in the host's local defense response to bacteria. Furthermore, the data allude to a conservation of this host response in evolution and suggest that a similar inducible pathway of host defense is prevalent at mucosal surfaces of mammals.

Analysis of lipopolysaccharide-response genes in B-lineage cells demonstrates that they can have differentiation stage-restricted expression and contain SH2 domains.

Bacterial lipopolysaccharide (LPS) is a potent stimulator of B-cell activation, proliferation, and differentiation. We examined the genetic response of B-lineage cells to LPS via trapping of expressed genes with a gene-trap retrovirus. This analysis showed that expression of only a small fraction of genes is altered during LPS stimulation of B-lineage cells. Isolation of the cellular portion of the trapped LPS-response genes via 5' RACE (rapid amplification of cDNA ends) cloning identified novel genes for all the cloned loci. These novel LPS-response genes were also found to have differentiation stage-restricted expression within the B-lymphoid lineage. That LPS-response genes in B cells also have differentiation stage-restricted expression suggests that these genes may be involved in the control of B-cell function and differentiation, since the known members of this class of genes have frequently been found to play a role in the function and differentiation of B-lineage cells. The isolation of novel members of this class of genes, including a gene that contains a putative SH2 domain, will further increase our understanding of the molecular events involved in the control of B-cell differentiation and function.

CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency.

Tumor necrosis factor (TNF) mediates a wide variety of disease states including septic shock, acute and chronic inflammation, and cachexia. Recently, a multivalent guanylhydrazone (CNI-1493) developed as an inhibitor of macrophage activation was shown to suppress TNF production and protect against tissue inflammation and endotoxin lethality [Bianchi, M., Ulrich, P., Bloom, O., Meistrell, M., Zimmerman, G. A., Schmidtmayerova, H., Bukrinsky, M., Donnelley, T., Bucala, R., Sherry, B., Manogue, K. R., Tortolani, A. J., Cerami, A. & Tracey, K. J. (1995) Mol. Med. 1, 254-266, and Bianchi, M., Bloom, O., Raabe, T., Cohen, P. S., Chesney, J., Sherry, B., Schmidtmayerova, H., Zhang, X., Bukrinsky, M., Ulrich, P., Cerami, A. & Tracey, J. (1996) J. Exp. Med., in press]. We have now elucidated the mechanism by which CNI-1493 inhibits macrophage TNF synthesis and show here that it acts through suppression of TNF translation efficiency. CNI-1493 blocked neither the lipopolysaccharide (LPS)-induced increases in the expression of TNF mRNA nor the translocation of nuclear factor NF-kappa B to the nucleus in macrophages activated by 15 min of LPS stimulation, indicating that CNI-1493 does not interfere with early NF-kappa B-mediated transcriptional regulation of TNF. However, synthesis of the 26-kDa membrane form of TNF was effectively blocked by CNI-1493. Further evidence for the translational suppression of TNF is given by experiments using chloram-phenicol acetyltransferase (CAT) constructs containing elements of the TNF gene that are involved in TNF translational regulation. Both the 5' and 3' untranslated regions of the TNF gene were required to elicit maximal translational suppression by CNI-1493. Identification of the molecular target through which CNI-1493 inhibits TNF translation should provide insight into the regulation of macrophage activation and mechanisms of inflammation.

Activation of c-Jun N-terminal kinase in bacterial lipopolysaccharide-stimulated macrophages.

Activation of macrophages by bacterial lipopolysaccharide (LPS) induces transcription of genes that encode for proinflammatory regulators of the immune response. Previous work has suggested that activation of the transcription factor activator protein 1 (AP-1) is one LPS-induced event that mediates this response. Consistent with this notion, we found that LPS stimulated AP-1-mediated transcription of a transfected reporter gene in the murine macrophage cell line RAW 264.7. As AP-1 activity is regulated in part by activation of the c-Jun N-terminal kinase (JNK), which phosphorylates and subsequently increases the transcriptional activity of c-Jun, we examined whether LPS treatment of macrophages resulted in activation of this kinase. LPS treatment of RAW 264.7 cells, murine bone marrow-derived macrophages, and the human monocyte cell line THP-1 resulted in rapid activation of the p46 and p54 isoforms of JNK. Treatment with wild-type and rough mutant forms of LPS and synthetic lipid A resulted in JNK activation, while pretreatment with the tyrosine kinase inhibitor herbimycin A inhibited this response. Binding of LPS-LPS binding protein (LBP) complexes to CD14, a surface receptor that mediates many LPS responses, was found to be crucial, as pretreatment of THP-1 cells with the monoclonal antibody 60b, which blocks this binding, inhibited JNK activation. These results suggest that LPS activation of JNK in monocyte/macrophage cells is a CD14- and protein tyrosine phosphorylation-dependent event that may mediate the early activation of AP-1 in regulating LPS-triggered gene induction.

Arrest of endotoxin-induced hypotension by transforming growth factor beta1.

Septic shock is a cytokine-mediated process typically caused by a severe underlying infection. Toxins generated by the infecting organism trigger a cascade of events leading to hypotension, to multiple organ system failure, and frequently to death. Beyond supportive care, no effective therapy is available for the treatment of septic shock. Nitric oxide (NO) is a potent vasodilator generated late in the sepsis pathway leading to hypotension; therefore, NO represents a potential target for therapy. We have previously demonstrated that transforming growth factor (TGF) beta1 inhibits inducible NO synthase (iNOS) mRNA and NO production in vascular smooth muscle cells after its induction by cytokines critical in the sepsis cascade. Thus, we hypothesized that TGF-beta1 may inhibit iNOS gene expression in vivo and be beneficial in the treatment of septic shock. In a conscious rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS), TGF-beta1 markedly reduced iNOS mRNA and protein levels in several organs. In contrast, TGF-beta1 did not decrease endothelium-derived constitutive NOS mRNA in organs of rats receiving LPS. We also performed studies in anesthetized rats to evaluate the effect of TGF-beta1 on the hemodynamic compromise of septic shock; after an initial 25% decrease in mean arterial pressure, TGF-beta1 arrested LPS-induced hypotension and decreased mortality. A decrease in iNOS mRNA and protein levels in vascular smooth muscle cells was demonstrated by in situ hybridization and NADPH diaphorase staining in rats treated with TGF-beta1. Thus these studies suggest that TGF-beta1 inhibits iNOS in vivo and that TGF-beta1 may be of future benefit in the therapy of septic shock.

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

CD14 enhances cellular responses to endotoxin without imparting ligand-specific recognition.

Binding of the lipid A portion of bacterial lipopolysaccharide (LPS) to leukocyte CD14 activates phagocytes and initiates the septic shock syndrome. Two lipid A analogs, lipid IVA and Rhodobacter sphaeroides lipid A (RSLA), have been described as LPS-receptor antagonists when tested with human phagocytes. In contrast, lipid IVA activated murine phagocytes, whereas RSLA was an LPS antagonist. Thus, these compounds displayed a species-specific pharmacology. To determine whether the species specificity of these LPS antagonists occurred as a result of interactions with CD14, the effects of lipid IVA and RSLA were examined by using human, mouse, and hamster cell lines transfected with murine or human CD14 cDNA expression vectors. These transfectants displayed sensitivities to lipid IVA and RSLA that reflected the sensitivities of macrophages of similar genotype (species) and were independent of the source of CD14 cDNA. For example, hamster macrophages and hamster fibroblasts transfected with either mouse or human-derived CD14 cDNA responded to lipid IVA and RSLA as LPS mimetics. Similarly, lipid IVA and RSLA acted as LPS antagonists in human phagocytes and human fibrosarcoma cells transfected with either mouse or human-derived CD14 cDNA. Therefore, the target of these LPS antagonists, which is encoded in the genomes of these cells, is distinct from CD14. Although the expression of CD14 is required for macrophage-like sensitivity to LPS, CD14 cannot discriminate between the lipid A moieties of these agents. We hypothesize that the target of the LPS antagonists is a lipid A recognition protein which functions as a signaling receptor that is triggered after interaction with CD14-bound LPS.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.

Inflammation-induced recombinant protein expression in vivo using promoters from acute-phase protein genes.

We report that promoters for two murine acute-phase protein (APP) genes, complement factor 3 (C3) and serum amyloid A3 (SAA3), can increase recombinant protein expression in response to inflammatory stimuli in vivo. To deliver APP promoter-luciferase reporter gene constructs to the liver, where most endogenous APP synthesis occurs, we introduced them into a nonreplicating adenovirus vector and injected the purified viruses intravenously into mice. When compared with the low levels of basal luciferase expression observed prior to inflammatory challenge, markedly increased expression from the C3 promoter was detected in liver in response to both lipopolysaccharide (LPS) and turpentine, and lower-level inducible expression was also found in lung. In contrast, expression from the SAA3 promoter was found only in liver and was much more responsive to LPS than to turpentine. After LPS challenge, hepatic luciferase expression increased rapidly and in proportion to the LPS dose. Use of cytokine-inducible promoters in gene transfer vectors may make it possible to produce antiinflammatory proteins in vivo in direct relationship to the intensity and duration of an individual's inflammatory response. By providing endogenously controlled production of recombinant antiinflammatory proteins, this approach might limit the severity of the inflammatory response without interfering with the beneficial components of host defense and immunity.

Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat.

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.