IL-1 receptor accessory protein is an essential component of the IL-1 receptor.

The recently described IL-1R accessory protein (IL-1R AcP) interacts with IL-1beta and the IL-1 type-IR (IL-1RI), but an essential requirement for IL-1R AcP in IL-1 signaling in vitro has not been established and its role in vivo has not been examined. In this study, IL-1R AcP-deficient mice and fibroblasts were produced and characterized. All IL-1 agonists bound to IL-1R AcP-deficient cells through the type I IL-1R, but failed to activate gene expression through either the nuclear factor-kappaB or AP-1-dependent signaling pathways. Absence of IL-1R AcP differentially affected the affinity for IL-1 ligands. IL-1R AcP-deficient fibroblasts bound murine IL-1alpha and human IL-1R antagonist protein (IL-1Ra) with only moderately reduced affinity when compared with wild-type cells, whereas murine IL-1beta affinity was reduced by 70-fold. IL-1 also failed to produce a biologic response in vivo in IL-1R AcP-deficient mice. These data demonstrate that a type I IL-1R/IL-1R AcP complex is required for signaling by all IL-1 agonists and for high affinity binding by IL-1beta. Finally, IL-1R AcP is an essential signal transducing component of the functional IL-1R and should represent a novel target for blocking IL-1 function in human disease.

SEK1/MKK4 is required for maintenance of a normal peripheral lymphoid compartment but not for lymphocyte development.

SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate control of cellular death and proliferation. In lymphocytes, the SAPK pathway has been implicated in signaling from antigen, costimulatory, and death receptors; SEK1, which directly activates SAPK, is required for early embryonic development and has also been reported to be essential for normal lymphocyte development. In contrast to the latter findings, we have used RAG-2-deficient blastocyst complementation to show that SEK1-deficient embryonic stem cells support unimpaired T and B lymphocyte development. Moreover, mature SEK1-deficient lymphocytes are capable of SAPK activation. Surprisingly, however, aging SEK1-deficient chimeric mice frequently develop lymphadenopathy and polyclonal B and T cell expansions. Thus, SEK1 is not required for lymphocyte development, but is required for maintaining peripheral lymphoid homeostasis.

SEK1 deficiency reveals mitogen-activated protein kinase cascade crossregulation and leads to abnormal hepatogenesis.

SEK1 (MKK4/JNKK) is a mitogen-activated protein kinase activator that has been shown to participate in vitro in two stress-activated cascades terminating with the SAPK and p38 kinases. To define the role of SEK1 in vivo, we studied stress-induced signaling in SEK1(-/-) embryonic stem and fibroblast cells and evaluated the phenotype of SEK1(-/-) mouse embryos during development. Studies of SEK1(-/-) embryonic stem cells demonstrated defects in stimulated SAPK phosphorylation but not in the phosphorylation of p38 kinase. In contrast, SEK1(-/-) fibroblasts exhibited defects in both SAPK and p38 phosphorylation, demonstrating that crosstalk exists between the stress-activated cascades. Tumor necrosis factor alpha and interleukin 1 stimulation of both stress-activated cascades are severely affected in the SEK1(-/-) fibroblast cells. SEK1 deficiency leads to embryonic lethality after embryonic day 12.5 and is associated with abnormal liver development. This phenotype is similar to c-jun null mouse embryos and suggests that SEK1 is required for phosphorylation and activation of c-jun during the organo-genesis of the liver.

Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice.

PURPOSE: During long-term treatment of various malignant or viral diseases with IFN-alpha up to 20% of patients develop anti-IFN-alpha antibodies for as yet unknown reasons. METHODS: To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN-alpha antibodies inducing factors was studied. RESULTS: The model revealed that both a higher frequency of injections and a higher dosage of IFN-alpha were more immunogenic and that the route of administration affected the antibody response to IFN-alpha. The intrinsic immunostimulatory activity of IFN-alpha itself also enhanced the immune response. IFN-alpha protein aggregates (IFN-alpha-IFN-alpha and human serum albumin (HSA)-IFN-alpha aggregates), which were recently identified in all marketed IFN-alpha products, were significantly more immunogenic than IFN-alpha monomers. These aggregates broke the tolerance against human IFN-alpha monomers in human IFN-alpha transgenic mice. CONCLUSIONS: Based on these animal studies it is proposed that the immune response to IFN-alpha in humans is most probably elicited by a combination of several factors among which IFN-alpha protein aggregates seem to play a key role.

Cre-mediated generation of a VCAM-1 null allele in transgenic mice.

A conditional null allele for VCAM-1 was generated in mice through a one step ES cell selection procedure by flanking the proximal promoter and exons 1 and 2 with loxP sites. The ES cells were used to create chimeric mice, which were then used to produce mice homozygous for the VCAM-1 conditional null, or floxed allele. Although the PGKneo cassette was retained in the promoter, the homozygous mice produced levels of VCAM-1 transcripts similar to that seen in wild-type mice. Homozygous VCAMflox/flox mice were mated to transgenic lines of mice expressing the cre gene under control of the murine platelet endothelial cell adhesion molecule-1 (PECAM-1) promoter. Surprisingly, the VCAMflox allele in all tissues examined from mice that inherited the cre-transgene had underwent complete excision of the floxed VCAM-1 sequences. The 'deleted' VCAM-1 allele (VCAMdel) was stably inherited, even in those mice that did not inherit the cre transgene, indicating the recombination occurs at an early stage of development prior to germ cell development. Thus the cre mice can be used for ubiquitous gene rearrangement in vivo. The data also suggest a novel simplified strategy for using the Cre/loxP system in vivo, in which a single ES cell and line of mice can be used to create mice carrying either a null or conditional null allele.

Heterogeneity of expression of E- and P-selectins in vivo.

A novel technique involving radiolabeled monoclonal antibodies was used to characterize and compare the expression of E- and P-selectin on unstimulated, histamine-challenged, and endotoxin-challenged endothelial cells in various tissues of the mouse. Under unstimulated conditions, E-selectin was absent in all organs, but significant expression of P-selectin was observed in several organs. Histamine induced a rapid time-dependent upregulation of P-selectin, with the largest responses observed in mesentery and lung. Significant fold elevations in P-selectin expression occurred as early as 5 minutes after the histamine injection and remained elevated up to 1 hour. Histamine-induced P-selectin upregulation was inhibited by the H1 receptor antagonist diphenhydramine, whereas the H2 receptor antagonist cimetidine had no effect. Endotoxin (lipopolysaccharide [LPS]) also induced a time-dependent expression of P-selectin that reached a maximum between 4 and 8 hours after endotoxin administration. LPS-induced upregulation of P-selectin was greatest in heart and stomach, which exhibited insignificant constitutive expression of P-selectin. LPS also induced a time-dependent upregulation of E-selectin, with maximal expression occurring 3 to 5 hours after intraperitoneal administration. The lung and small intestine exhibited the largest responses to LPS challenge. Histamine administration did not affect E-selectin expression in any tissue. E- and P-selectin-deficient mice were used to test the specificity of monoclonal antibody binding in unstimulated, histamine-challenged, and LPS-stimulated tissues. Vascular binding of the radiolabeled E-selectin and P-selectin monoclonal antibodies was not observed in the respective deficient mice. These findings suggest that P-selectin is constitutively expressed on vascular endothelium in some tissues of the mouse and that there are significant regional differences in the magnitude and time course of histamine- and endotoxin-induced P-selectin expression. In contrast, E-selectin appears to be absent on unstimulated vascular endothelium but is upregulated within 3 hours after the administration of endotoxin in most tissues.

Reproduction in mice lacking a functional type 1 IL-1 receptor.

Growth factors and cytokines have been identified in having critical roles at mediating maternal-fetal interactions during pregnancy, with interleukin-1 being a recently implicated factor. Previous experiments indicated that repeated intraperitoneal injections of the Il-1 receptor antagonist (Il-1Ra), which inhibits binding of interleukin-1 (Il-1) to the type 1 Il-1 receptor (Il-1Rt1) blocks blastocyst implantation in superovulated mice. To gain a greater insight into the role of Il-1 receptor in implantation, we analyzed the reproduction of mice deficient for the Il-1Rt1. Our results show that mice lacking this receptor do not exhibit any profound alterations in their reproduction, apart from a slight reduction in mean litter size. Furthermore, repeated intraperitoneal injections of either IL-1Ra or the monoclonal antibody 35F5, which also blocks ligand binding to the Il-1Rt1, did not affect embryo implantation in either wild type and Il-1 receptor deficient mice.

Identification and characterization of a novel surface antigen gene induced in mast cells activated through the high affinity IgE receptor.

In an effort to isolate novel genes involved in inflammation and/or mast cell activation, we have used a combination of differential screening and subtractive hybridization to isolate genes whose expression are induced upon activation of a transformed rat mast cell line. One of the isolated clones, pMCA-32, contained an open reading frame of 278 amino acids that included a putative hydrophobic transmembrane domain, a cysteine rich Ig-like extracellular domain, and a cytoplasmic domain containing three consensus SH2-domain phosphotyrosine binding sites. The MCA-32 gene is also highly conserved between rat and mouse, with the two coding regions being 73% identical. Although the MCA-32 coding region did not contain an obvious signal peptide, MCA-32 protein was detected on the surface of rat mast cells, and the cloned cDNA produced a cell surface protein when expressed in COS-7 cells. MCA-32 RNA from both mouse and rat undergoes alternative splicing, producing an mRNA containing an in-frame deletion of the TM domain, suggesting that a form of MCA-32 protein may be secreted. MCA-32 mRNA expression was up-regulated upon activation of RBL-2H3 cells and was highly abundant in primary peritoneal mast cells. Expression of MCA-32 RNA was only observed in primary and transformed mast cells from rat, while in the mouse MCA-32, RNA was also produced in significant amounts by a number of transformed monocyte cell lines. Thus, MCA-32 is a novel surface protein whose structure and expression suggest roles in the development and/or activation of mast cells and monocytes.

Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice.

VCAM-1 is a cytokine-inducible cell surface protein capable of mediating adhesion to leukocytes expressing alpha 4 integrins. Mice deficient in VCAM-1 expression were produced by targeted homologous recombination in ES cells. VCAM-1-deficient embryos were not viable and exhibited either of two distinct phenotypes. Approximately half of the embryos died before embryonic day 11.5 and exhibited a severe defect in placental development in which the allantois failed to fuse with the chorion. The remaining VCAM-1-deficient embryos survived to embryonic day 11.5-12.5 and displayed several abnormalities in their developing hearts including a reduction of the compact layer of the ventricular myocardium and intraventricular septum. The hearts also contained significant amounts of blood in the pericardial space and lacked an epicardium. alpha 4 and VCAM-1 were found to be expressed in wild-type embryos in a reciprocal fashion in the chorion and allantois and in the epicardium and the underlying myocardium, although VCAM-1 was expressed in the intraventricular septum in the absence of adjacent alpha 4-expressing cells. These data suggest important roles for VCAM-1 and alpha 4 in the development of the placenta and the heart.

Murine vascular cell adhesion molecule-1 (VCAM-1) proteins encoded by alternatively spliced mRNAs are differentially targeted in polarized cells.

VCAM-1 is an immunoglobulin (Ig) superfamily member expressed in endothelial cells that mediates adhesion to a variety of leukocytes in a VLA-4 dependent manner. In the mouse, two distinct forms of VCAM are produced. One form, VCAMTM, contains seven Ig domains followed by a single transmembrane region and a short cytoplasmic domain. A second form, VCAMGPI, which is preferentially induced by cytokines and LPS, contains only the first three Ig domains and is attached to the cell surface via a glycosylphosphafidylinositol (GPI) anchor. Both vascular and nonvascular expression of VCAM have been reported in a variety of normal and pathological settings. One possible role for the two VCAM isoforms is to allow for the targeted localization of VCAM to specific cell surface domains of polarized cells. This may be particularly relevant since VCAM is known to be expressed by two different polarized cell types, namely endothelial cells and kidney epithelial cells. In this study, MDCK cells permanently expressing either VCAMTM or VCAMGPI were established and used to examine the targeting of VCAM proteins to different polarized surface domains. VCAMTM was primarily located on the basolateral surface while VCAMGPI was located on the apical surface of polarized MDCK cells. Data is also presented that demonstrates that polarized expression is reversed in endothelial cells where VCAMTM was observed primarily on the apical surface. The differential localization of VCAM isoforms on the cell surface has direct implications for the ability of VCAM to mediate cell adhesion and transmigration.

Characterization of E-selectin-deficient mice: demonstration of overlapping function of the endothelial selectins.

The initial rolling interaction of leukocytes with the blood vessel wall during leukocyte trafficking has been postulated to rely on members of the selectin family of adhesion molecules. Two selectins, E-selectin and P-selectin, have been identified that are expressed on activated endothelial cells. Mice deficient in E-selectin expression have been produced in order to examine the role of this selectin in leukocyte trafficking. Mice homozygous for an E-selectin null mutation were viable and exhibited no obvious developmental alterations. E-selectin-deficient mice displayed no significant change in the trafficking of neutrophils in several models of inflammation. However, blocking both endothelial selectins by treatment of the E-selectin-deficient animals with an anti-murine P-selectin antibody, 5H1, significantly inhibited neutrophil emigration in two distinct models of inflammation. While neutrophil accumulation at early times during thioglycollate-induced peritonitis was dependent on P-selectin, neutrophil accumulation at later time points was blocked by 5H1 only in E-selectin-deficient mice but not in wild-type mice. Similarly, edema as well as leukocyte accumulation in a model of delayed-type hypersensitivity in the skin was almost completely prevented by blockade of P-selectin function with 5H1 in the E-selectin-deficient mice while the same treatment had no effect in wild-type mice. These data demonstrate that the majority of neutrophil migration in both models requires an endothelial selectin but that E-selectin and P-selectin are functionally redundant. These data have important implications in the use of selectin antagonists in the treatment of inflammatory disease.

Cytokine induction of an alternatively spliced murine vascular cell adhesion molecule (VCAM) mRNA encoding a glycosylphosphatidylinositol-anchored VCAM protein.

VCAM-1 is an immunoglobulin superfamily member that mediates adhesion of a variety of leukocytes to endothelial cells. VCAM expression has been associated with a variety of disease states and has been implicated in a number of normal processes. The predominant form of VCAM produced in human endothelial cells is a transmembrane protein containing seven immunoglobulin domains. In this study the murine VCAM gene has been characterized to allow the function(s) of VCAM to be studied in a small genetically accessible animal. While expression of an mRNA encoding a seven-immunoglobulin-domain transmembrane VCAM protein was seen in most tissues, the predominant change in VCAM expression upon interleukin 1 beta treatment was the induction of an alternatively spliced VCAM mRNA containing only the first three immunoglobulin domains. This message encodes a glycosylphosphatidylinositol (GPI)-anchored form of VCAM, VCAMGPI. VCAMGPI was efficiently cleaved from the cell surface by phosphatidylinositol-specific phospholipase C, mediated adhesion to leukocytes in a very late antigen 4-dependent manner, and was produced by mouse endothelial cell lines in culture. These data demonstrate that alternate forms of VCAM are produced under different physiological conditions and suggest that VCAMGPI may have a distinct role in inflammatory processes.

A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside.

LAP267 is a lacI activator protein (LAP) containing an insertion of the transcriptional activation domain of the herpes simplex virus virion protein 16 within the inducer-binding and dimerization domain of the lac repressor protein. LAP267 strongly induces expression in a conditional manner from a minimal simian virus 40 early promoter linked to lac operator sequences. LAP267 is temperature-sensitive, activating expression at 32 degrees C but not at 39.5 degrees C. It is allosterically regulated in a manner opposite that of wild-type lac repressor, in that LAP267 activity is rescued at the nonpermissive temperature by isopropyl beta-D-thiogalactopyranoside (IPTG). Stable mouse cell lines containing both the LAP267 gene and a LAP-inducible chloramphenicol acetyltransferase (CAT) reporter gene were readily established and exhibited up to a 1200-fold increase in CAT activity within 24 hr upon addition of IPTG. Thus, LAP267 is a powerful inducible switch in mammalian cells, imparting a regulatory stringency similar to that observed with lac repressor in Escherichia coli.

Expression of the herpes simplex virus 1 alpha transinducing factor (VP16) does not induce reactivation of latent virus or prevent the establishment of latency in mice.

A feature of the cascade regulation of herpes simplex virus 1 gene expression in productive infection is that the first genes to be expressed, the alpha genes, are transactivated by a structural component of the virion designated as the alpha transinducing factor (alpha TIF). In this study, we have tested the hypothesis that latent infection of sensory neurons results from the failure of alpha TIF, a tegument protein, to be transported from the nerve endings to the nucleus of the sensory neuron. Two viruses were constructed. The first recombinant virus (R6003) contained a second copy of the alpha TIF gene placed under the control of a metallothionein promoter. The second recombinant virus (R6004) is identical to R6003 except for the presence of a stop codon inserted at amino acid 70 of the second alpha TIF gene. The metallothionein promoter inserted into the viral genome was shown to be expressed, and alpha TIF mRNA was detected by in situ hybridization of sections of trigeminal ganglia of mice infected with R6003, both untreated and those given cadmium injections. In all experiments, there were no significant differences in the recovery of latent virus from mice infected with R6003 or R6004, whether injected with cadmium or not. Cadmium administration at the time of infection and at intervals thereafter did not preclude establishment of latency. In another series of experiments, transgenic mice expressing the metallothionein-driven alpha TIF did not differ from nontransgenic siblings with respect to the incidence of latent virus in trigeminal ganglia. We conclude that the absence of alpha TIF cannot alone account for the establishment of latency.

Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.

The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome is described. In this construct SV40 regulatory sequences, including the early promoter/enhancers and origin of DNA replication, were substituted for the AAV p5 promoter, which normally controls expression of the AAV rep gene. The hybrid genome was phenotypically indistinguishable from wild-type AAV in human cells in the presence or absence of helper virus. Upon transfection into cos-7 cells, which constitutively produced the SV40 tumor antigen, the genome replicated as a plasmid when the SV40 origin was used, although with a low efficiency compared with that of a non-AAV/SV40 replicon. The low level of replication was due to an inhibitory effect of an AAV rep gene product and was specific for replicons containing AAV sequences. Target AAV sequences required for inhibition by rep appeared to reside in the terminal repetitions since deletion of these sequences allowed efficient replication in the presence of the rep gene. The possible role for negative autoregulation of AAV DNA replication in latent infection and helper-dependent replication by AAV is discussed.

Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes.

In this paper we report that adeno-associated virus (AAV) genomes inhibit stable transformation by several dominant selectable marker genes upon cotransfection into mouse tissue culture cells. Cotransfection of AAV genomes also inhibited the expression of pSV2cat in transient assays. In both cases, the inhibitory effect was independent of AAV DNA replication but required the AAV p5 and p19 genes, which encode proteins required for AAV DNA replication and regulation of AAV gene expression. Finally, addition of a cloned E4 gene in the transfection experiments partially blocked the AAV-mediated inhibitory activities.