Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses.

Mammals mount a rapid inflammatory response to gram-negative bacteria by recognizing lipopolysaccharide (LPS, endotoxin). LPS binds to CD14, and the resulting LPS-CD14 complex induces synthesis of cytokines and up-regulation of adhesion molecules in a variety of cell types. Gram-positive bacteria provoke a very similar inflammatory response, but the molecules that provoke innate responses to these bacteria have not been defined. Here we show that protein-free, phenol extracts of Staphylococcus aureus contain a minor component that stimulates adhesion of neutrophils and cytokine production in monocytes and in the astrocytoma cell line, U373. Responses to this component do not absolutely require CD14, but addition of soluble CD14 enhances sensitivity of U373 cells by up to 100-fold, and blocking CD14 on monocytes decreases sensitivity nearly 1,000-fold. Deletion of residues 57-64 of CD14, which are required for responses to LPS, also eliminates CD14-dependent responses to S. aureus molecules. The stimulatory component of S. aureus binds CD14 and blocks binding of radioactive LPS. Unlike LPS, the activity of S. aureus molecules was neither enhanced by LPS binding protein nor inhibited by bactericidal/permeability increasing protein. The active factor in extracts of S. aureus is also structurally and functionally distinct from the abundant species known as lipoteichoic acid (LTA). Cell-stimulating activity fractionates differently from LTA on a reverse-phase column, pure LTA fails to stimulate cells, and LTA antagonizes the action of LPS in assays of IL-6 production. These studies suggest that mammals may use CD14 in innate responses to both gram-negative and gram-positive bacteria, and that gram-positive bacteria may contain an apparently unique, CD14-binding species that initiates cellular responses.

A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6.

We have cloned the promoter for the human third component of complement (C3) gene and have identified sequences involved in its regulation during the acute-phase response. A construct linking 199 bp of the C3 promoter to the firefly luciferase gene was found to be very responsive to interleukin-1 (IL-1) and modestly responsive to interleukin-6 (IL-6) by transfection analysis in the human hepatoma line Hep3B2. Simultaneous treatment with the two cytokines showed a strong synergy between the actions of the two molecules. A 58-bp fragment (-127 to -70 bp) was shown by 5' and 3' deletional mutagenesis to contain cis-acting elements that mediated both the IL-1 response and the IL-1-plus-IL-6 synergistic response of this promoter. When coupled to a heterologous promoter, this fragment enabled the synergistic induction by IL-1 plus IL-6. Sequences homologous to the palindrome ACATTGCACAATCT, which mediates the induction of the IL-6 gene by IL-1 (S. Akira, H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, and T. Kishimoto, EMBO J. 9:1897-1906, 1990), and the core sequence of the IL-6-responsive element of the rat alpha 2-macroglobulin gene (CTGGGA; M. Hattori, L. J. Abraham, W. Northemann, and G. H. Fey, Proc. Natl. Acad. Sci. USA 87:2364-2368, 1990) are contained within this fragment in immediate juxtaposition and partially overlapping. Site-directed mutagenesis within this homology region drastically reduced the inducibility of the C3 promoter by either cytokine. DNase I footprinting analysis defined a binding site for the transcription factor CCAAT/enhancer-binding protein (C/EBP), which included the IL-1-responsive element-like sequence. No differences were seen between the footprints generated by using extracts from unstimulated and IL-1-stimulated Hep3B2 cells. However, gel retardation analyses revealed two IL-1-specific bands. The data suggest that the induction by IL-1 is mediated by a factor belonging to the family of C/EBP-related proteins.

Cloning and characterization of a specific receptor for mouse oncostatin M.

Oncostatin M (OSM) is a member of a family of cytokines that includes ciliary neurotrophic factor, interleukin-6, interleukin-11, cardiotrophin-1, and leukemia inhibitory factor (LIF). The receptors for these cytokines consist of a common signaling subunit, gp130, to which other subunits are added to modify ligand specificity. We report here the isolation and characterization of a cDNA encoding a subunit of the mouse OSM receptor. In NIH 3T3 cells (which endogenously express gp130, LIF receptor beta [LIFRbeta], and the protein product, c12, of the cDNA described here), mouse LIF, human LIF, and human OSM signaled through receptors containing the LIFRbeta and gp130 but not through the mouse OSM receptor. Mouse OSM, however, signaled only through a c12-gp130 complex; it did not use the LIF receptor. Binding studies demonstrated that mouse OSM associated directly with either the c12 protein or gp130. These data highlight the species-specific differences in receptor utilization and signal transduction between mouse and human OSM. In mouse cells, only mouse OSM is capable of activating the mouse OSM receptor; human OSM instead activates the LIF receptor. Therefore, these data suggest that all previous studies with human OSM in mouse systems did not elucidate the biology of OSM but, rather, reflected the biological actions of LIF.

Molecular characterization of mouse and rat CPP32 beta gene encoding a cysteine protease resembling interleukin-1 beta converting enzyme and CED-3.

Interleukin-1 beta converting enzyme (ICE) defines a new class of mammalian cysteine protease that shares strong homology with the Caenorhabditis elegans death gene ced-3. Both ICE and CED-3, when introduced into cultured cells, induce apoptosis, indicating that this type of cysteine protease may play an important role in the process of programmed cell death. Here, we report the cloning of a mouse and rat gene encoding a novel cysteine protease. The putative proteins encoded by these cDNAs contain the conserved sequence (QACRG) necessary for covalent linkage to the substrate as well as the three amino acids responsible for substrate binding and catalysis in ICE. Amino acid sequence analysis indicates that this rodent cysteine protease is the homolog of human CPP32 beta. Mouse CPP32 beta mRNA is highly expressed in spleen, and to a lesser degree in brain, lung, liver, and kidney. The mouse CPP32 beta genomic locus spans a region of approximately 20 kb, including seven exons and six introns. Mouse interspecific backcross mapping allowed localization of CPP32 beta to the central region of mouse chromosome 8, linked to Scvr, Lpl, Jund1 and Mlr.

A caspase-resistant form of Bcl-X(L), but not wild type Bcl-X(L), promotes clonogenic survival after ionizing radiation.

Bcl-2 and Bcl-X(L) belong to a family of proteins overexpressed in a variety of human cancers which inhibit apoptosis in response to a number of stimuli including chemotherapeutic agents and ionizing radiation. To better understand the role of these polypeptides in modulating the response of cancer cells to ionizing radiation we used cell lines that were engineered to overexpress the two polypeptides. Although Bcl-2 and Bcl-X(L) overexpression resulted in inhibition of radiation-induced apoptosis, it did not result in enhanced clonogenic survival. Consistent with this was the observation that Bcl-2 and Bcl-X(L) protected cells from DNA fragmentation, loss of mitochondrial membrane potential, and caspase activation for up to 72 hours after irradiation. Beyond 72 hours, there was a rapid loss in the ability of Bcl-2 and Bcl-X(L) to inhibit these markers of apoptosis. When Bcl-X(L) was analyzed at 72 hours after irradiation and beyond, a rapid accumulation of a 16-kDa form of Bcl-X(L) was observed. To test the hypothesis that cleavage of the 29-kDa form of Bcl-X(L) by caspases to a 16-kDa polypeptide results in its inability to inhibit apoptosis beyond 72 hours, we constructed a cell line that overexpressed a caspase-resistant form of Bcl-X(L) (Bcl-X(L)-deltaloop). Cells overexpressing Bcl-X(L)-deltaloop were resistant to apoptosis beyond 72 hours after irradiation and did not contain the 16-kDa form at these time points. In addition, Bcl-X(L)-deltaloop overexpression resulted in enhanced clonogenic survival compared with control or Bcl-X(L) overexpressing cells. These results provide a molecular basis for the observation that expression of Bcl-2 or Bcl-X(L) is not a prognostic marker of tumor response to cancer therapy.

DNA binding by C/EBP proteins correlates with hepatocyte proliferation.

The leucine zipper transcription factors C/EBP alpha and C/EBP beta exhibit growth-related variations of expression and DNA binding during liver regeneration. We examined the expression of C/EBP proteins in relation to hepatocyte proliferation by studying their DNA-binding activity in primary mouse hepatocytes in vitro. Mouse hepatocytes were dissociated by collagenase perfusion and cultured in a serum-free, defined medium containing a variety of growth factors and hormones. Cell protein extracts were collected every 24 h for up to 10 d and examined for DNA-binding activity by gel retardation analysis using a C/EBP consensus sequence oligomer (bZIP). C/EBP alpha is the major bZIP-binding protein present in the dissociated cells prior to plating. With the culture conditions we employed, little or no binding of C/EBP proteins was observed in the first 24 to 48 h of cultivation. After 48 h, C/EBP beta binding activity was elevated relative to the level seen in freshly dissociated cells. In contrast, C/EBP alpha binding continued to be greatly reduced and no C/EBP delta binding was observed. C/EBP beta binding remained elevated for the duration of the experiment. Additional growth factor treatment (EGF, FGF, TGF alpha, and HGF) of the hepatocytes did not appreciably alter the pattern of C/EBP binding. However, TGF beta treatment, known to decrease hepatocyte proliferation, increased C/EBP beta binding activity earlier and more actively than in control cells. This study confirms a negative correlation between DNA binding by the C/EBP transactivator proteins and the proliferation of primary mouse hepatocytes in vitro.

Mice overexpressing murine oncostatin M (OSM) exhibit changes in hematopoietic and other organs that are distinct from those of mice overexpressing human OSM or bovine OSM.

Oncostatin M (OSM) and leukemia inhibitory factor (LIF) belong to the interleukin-6 family of cytokines. The authors' previous in vitro work demonstrated that in mouse cells mouse OSM (mOSM) signals through a heterodimeric receptor complex incorporating the mOSM-specific receptor mOSMRbeta while human OSM (hOSM) and bovine OSM (bOSM) use the mouse LIF receptor mLIFRbeta rather than mOSMRbeta. These in vitro data suggest that prior studies in mouse systems with hOSM or bOSM (the usual molecules used in early studies) reflect LIF rather than OSM biology. The current work assessed whether or not this divergence in actions among these three OSMs also occurs in vivo in mouse models. Adult female (C57BL/6J x DBA/2J) F(1) mice were engineered to stably overexpress mOSM, hOSM, or bOSM by retrovirus-mediated gene transfer (n = 10 or more per group). After 4 weeks, molecular and hematologic profiles and anatomic phenotypes in multiple organs were assessed by standard techniques. Animals overexpressing either hOSM or bOSM had an identical phenotype resembling that associated with LIF activation, including significant hematologic abnormalities (anemia, neutrophilia, lymphopenia, eosinopenia, and thrombocytosis); weight loss; profound enlargement (lymph node, spleen) and/or structural reorganization (lymph node, spleen, thymus) of lymphoid organs; and severe osteosclerosis. In contrast, mice overexpressing mOSM did not develop hematologic changes, weight loss, or osteosclerosis and exhibited more modest and anatomically distinct restructuring of lymphoid organs. These data indicate that activities imputed to OSM and the mOSMRbeta signaling pathway using in vitro and in vivo mouse experimental systems are unique to mOSM.

Participation of the transcription factor C/EBP delta in the acute-phase regulation of the human gene for complement component C3.

C3, the third component of complement, is critical in the host immune response in that it is involved in both the classical and alternative pathways of complement activation. We have previously shown that a region (bp -127 to -70) within the C3 promoter is indispensable for conferring interleukin 1 (IL-1) responsiveness to this gene. A sequence comparison reveals two CCAAT/enhancer binding protein (C/EBP) consensus sequences, basic DNA binding region and leucine zippers 1 and 2 (bZIP1 and bZIP2), within this region. Site-directed mutagenesis of the more 3' C/EBP site (bZIP1) in the C3 promoter significantly reduced the basal level of expression and the IL-1 responsiveness of the reporter gene, whereas mutation in the second, more 5', C/EBP consensus sequence (bZIP2) had a minimal effect on basal expression and IL-1 inducibility. Electrophoretic-mobility-shift assays, with and without antibodies to the different C/EBP proteins that "supershift" protein-DNA complexes, demonstrated that proteins binding at the 3' C/EBP site formed several complexes. Antibodies to C/EBP alpha supershifted the majority of complexes formed with extracts from control cells. Antibodies directed against C/EBP delta supershifted the major IL-1-inducible complexes. Western immunoblot analyses showed that the level of C/EBP delta protein was increased dramatically in the nuclei of Hep 3B2 cells after 4 h of IL-1 treatment. When Hep 3B2 cells were cotransfected with a C/EBP delta expression vector and a construct with a C3 promoter and a reporter gene, C/EBP delta was able to trans-activate the C3 promoter in an IL-1-responsive manner. The data strongly suggest that C/EBP delta is the major protein responsible for regulating the acute-phase expression of the human C3 gene.

Identification of a domain in soluble CD14 essential for lipopolysaccharide (LPS) signaling but not LPS binding.

CD14 is a 55-kDa glycoprotein that binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Monoclonal antibodies of CD14 such as 3C10 and MEM-18 are known to neutralize biological activity of CD14. Recently, it has been demonstrated that MEM-18 recognizes the LPS-binding site of CD14, between amino acids 57 and 64. It has also been shown that 3C10 recognizes a distinct epitope from that of MEM-18, indicating that 3C10 may yet define another functional domain of CD14. In order to identify the epitope for 3C10, we constructed a series of alanine substitution mutants of soluble CD14 (sCD14). BIAcore analyses showed that regions between amino acids 7 and 10 and between amino acids 11 and 14 are required for 3C10 binding. To assess the effect of altering the 3C10 epitope in CD14, we generated a stable cell line expressing a mutant sCD14 containing alanine substitutions in the region between amino acids 7 and 10, sCD14(7-10)A, and purified this protein to homogeneity. sCD14(7-10)A has impaired ability to mediate LPS-dependent IL-6 up-regulation in U373 cells, integrin activation in neutrophils, and NF-kappa B activation in U373 cells. Purified sCD14(7-10)A was, however, capable of forming a stable complex with LPS in an LPS binding protein-facilitated and LPS binding protein-independent fashion. The ability of sCD14(7-10)A to bind LPS was also demonstrated in assays in which excess sCD14(7-10)A inhibited LPS-mediated tumor necrosis factor-alpha production in whole blood and adhesion of polymorphonuclear leukocytes to fibrinogen. These data strongly suggest that a region recognized by neutralizing monoclonal antibody 3C10 contains a domain required for cellular signaling but not for LPS binding.

Soluble CD14 promotes LPS activation of CD14-deficient PNH monocytes and endothelial cells.

Bacterial lipopolysaccharide (LPS) initiates the cascade of inflammatory events that, in infected patients, often result in a lethal systemic inflammatory response known as the sepsis syndrome. We studied LPS-stimulated expression of tissue factor (TF) in human peripheral blood mononuclear cells (PBMCs) and cultured endothelial cells or tumor necrosis factor-alpha (TNF-alpha) in PBMCs. CD14, a PBMC membrane protein, is involved in LPS signaling and is also present as a soluble molecule in serum. CD14 is absent from endothelial cells and, in varying degrees, from monocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH). LPS stimulation of TF in normal monocytes was enhanced > 30-fold by serum at low concentrations of LPS (< or = 10 ng/ml). The serum dependence of endothelial cells was even more pronounced; a full response to LPS was not observed in endothelium under serum-free conditions, even with LPS concentrations as high as 100 ng/ml. To better define the role of CD14, CD14-deficient PBMCs from two patients with PNH were compared with normal PBMCs. Although less than 3% of PNH monocytes expressed CD14, LPS-induced synthesis of TF and TNF-alpha by PBMCs from PNH patients was inhibited by anti-CD14 antibodies. Because patient serum samples were found to contain soluble CD14, we sought to determine whether PNH monocytes might respond to LPS through an activation pathway dependent on soluble CD14. Recombinant soluble CD14 substituted for serum to enable LPS stimulation of endothelium, PNH PBMCs, and surprisingly, CD14-replete normal PBMCs. In addition, a truncated sCD14 containing the N-terminal 152 amino acids similarly enabled LPS stimulation of normal PBMCs. These data underscore the importance of soluble CD14 and suggest that CD14 present in serum enables LPS responses in PNH monocytes and endothelial cells and may even influence the effects of LPS in normal human phagocytes.

Identification and mapping of Casp7, a cysteine protease resembling CPP32 beta, interleukin-1 beta converting enzyme, and CED-3.

Cloning of interleukin-1 beta converting enzyme (ICE) and Caenorhabditis elegans death protein CED-3 revealed the structural and functional homology between these two proteases. It also suggested the involvement of ICE-like cysteine proteases in apoptosis. Several CED-3- and ICE-like cysteine proteases have been described, including Nedd2/Ich-1, CPP32 beta, Tx, ICErel3, and Mch2. We have previously described a mouse ortholog of cysteine protease CPP32 beta that shares strong homology with ICE and CED-3. Here, we describe the cloning of mouse and human Casp7, another member of this family of cysteine proteases. Mouse Casp7 encodes a putative 340-amino-acid polypeptide that contains all the known conserved residues required for protease function, including the QACRG sequence, aspartic acid residues for internal cleavage sites, and the residues required for substrate binding. Three RNA variants of human Casp7 were also cloned. Amino acid sequence analysis indicated that Casp7 shared high homology with CPP32 beta/Casp3 and Mch2/Casp6. Northern blot analysis demonstrated that a 2.6-kb Casp7 mRNA was expressed in various tissues except brain. Mouse interspecific backcross mapping allowed localization of Casp7 to the distal region of mouse chromosome 19, linked to Mxi1, Adra2a, and Aop1.

Identification of a lipopolysaccharide binding domain in CD14 between amino acids 57 and 64.

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. Recent limited proteolysis studies have implicated a region in CD14 between amino acids 57 and 64 as being involved in LPS interaction. To specifically assess the importance of this region with respect to LPS binding, we constructed a mutant sCD14 (sCD14 delta 57-64) lacking amino acids 57-64. sCD14 delta 57-64 was isolated from the serum-free conditioned medium of this cell line, and, in all assays, the purified protein failed to recognize LPS or enable LPS-dependent responses in cells. We also demonstrated that the region between amino acids 57 and 64 is required for binding of a neutralizing CD14 mAb, MEM-18. Native polyacrylamide gel electrophoresis assays were used to demonstrate that MEM-18 and LPS compete for the same binding site on CD14. These data strongly suggest that the region spanning amino acids 57-64 binds LPS and that formation of sCD14.LPS complex is required in order for sCD14-mediated responses to occur.

Soluble CD14 truncated at amino acid 152 binds lipopolysaccharide (LPS) and enables cellular response to LPS.

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. In order to identify the domains in CD14 required for function, we deleted increasing amounts of CD14 from the C terminus. Truncated CD14 cDNA sequences were transfected into COS-7 cells and serum-free conditioned medium was analyzed for mutant CD14 expression and bioactivity. Mutant CD14s containing as few as 152 amino acids were found to have activity equivalent to full-length sCD14. To further characterize the mutant CD14, we constructed a stable Chinese hamster ovary cell line expressing sCD14(1-152) and purified the protein to homogeneity. sCD14(1-152) bound radioactive LPS, enabled U373 cells to synthesize interleukin 6 in response to LPS, and enabled human neutrophils to respond to smooth LPS. In all of these assays, the behavior of sCD14(1-152) was quantitatively similar to full-length sCD14. We also found that two neutralizing anti-CD14 antibodies (3C10 and MEM-18) bound and neutralized sCD14(1-152). We conclude from these experiments that the N-terminal 152 amino acids of CD14 are sufficient to bind LPS and confer essentially wild-type bioactivity in vitro.

Chronic expression of murine flt3 ligand in mice results in increased circulating white blood cell levels and abnormal cellular infiltrates associated with splenic fibrosis.

The effect of chronic expression of flt3 ligand (FL) on in vivo hematopoiesis was studied. Retroviral vector-mediated gene transfer was used in a mouse model of bone marrow transplantation to enforce expression of mouse FL cDNA in hematopoietic tissues. As early as 2 weeks posttransplantation, peripheral blood white blood cell counts in FL-overexpressing recipients were significantly elevated compared with controls. With the exception of eosinophils, all nucleated cell lineages studied were similarly affected in these animals. Experimental animals also exhibited severe anemia and progressive loss of marrow-derived erythropoiesis. All of the FL-overexpressing animals, but none of the controls, died between 10 and 13 weeks posttransplantation. Upon histological examination, severe splenomegaly was noted, with progressive fibrosis and infiltration by abnormal lymphoreticular cells. Abnormal cell infiltration also occurred in other organ systems, including bone marrow and liver. In situ immunocytochemistry on liver sections showed that the cellular infiltrate was CD3+/NLDC145+/CD11c+, but B220- and F4/80-, suggestive of a mixed infiltrate of dendritic cells and activated T lymphocytes. Infiltration of splenic blood vessel perivascular spaces resulted in vascular compression and eventual occlusion, leading to splenic necrosis consistent with infarction. These results show that FL can affect both myeloid and lymphoid cell lineages in vivo and further demonstrate the potential toxicity of in vivo treatment with FL.