Antiviral activity of tumor necrosis factor (TNF) is mediated via p55 and p75 TNF receptors.

The antiviral nature of tumor necrosis factor (TNF) is generally well accepted. TNF appears to induce multiple antiviral mechanisms, and to synergize with interferon (IFN)-gamma in promoting antiviral activities. We infected TNF receptor (TNFR)-deficient mice with the virulent murine pathogen, ectromelia virus (EV), and observed that otherwise resistant mice were susceptible to lethal infection. To study the molecular basis of the antiviral action of TNF, mice were infected with a recombinant vaccinia virus encoding murine TNF (VV-HA-TNF). In normal mice, the replication of VV-HA-TNF was highly attenuated. In contrast, mice in which the TNFR type 1 (p55) or the TNFR type 2 (p75) were genetically disrupted showed a moderate defect in their capacity to clear the TNF-encoding virus. The contribution of both TNF receptors to the control of VV-HA-TNF was confirmed by the enhanced replication of VV-HA-TNF in mice deficient for both p55 and p75. These observations were corroborated by infecting TNFR-deficient mice with EV. For both infections, the p55 and p75 TNFRs were necessary to maintain normal levels of resistance. Thus, the antiviral activity of TNF is mediated via both TNFRs in vivo. Furthermore, these studies establish that TNF is an important component of the host response to a natural virus infection.

Distinct roles for signals relayed through the common cytokine receptor gamma chain and interleukin 7 receptor alpha chain in natural T cell development.

The commitment, differentiation, and expansion of mainstream alpha/beta T cells during ontogeny depend on the highly controlled interplay of signals relayed by cytokines through their receptors on progenitor cells. The role of cytokines in the development of natural killer (NK)1(+) natural T cells is less clearly understood. In an approach to define the role of cytokines in the commitment, differentiation, and expansion of NK1(+) T cells, their development was studied in common cytokine receptor gamma chain (gammac) and interleukin (IL)-7 receptor alpha (IL-7Ralpha)-deficient mice. These mutations block mainstream alpha/beta T cell ontogeny at an early prethymocyte stage. Natural T cells do not develop in gammac-deficient mice; they are absent in the thymus and peripheral lymphoid organs such as the liver and the spleen. In contrast, NK1(+) T cells develop in IL-7Ralpha-deficient mice in the thymus, and they are present in the liver and in the spleen. However, the absolute number of NK1(+) T cells in the thymus of IL-7Ralpha-deficient mice is reduced to approximately 10%, compared to natural T cell number in the wild-type thymus. Additional data revealed that NK1(+) T cell ontogeny is not impaired in IL-2- or IL-4-deficient mice, suggesting that neither IL-2, IL-4, nor IL-7 are required for their development. From these data, we conclude that commitment and/or differentiation to the NK1(+) natural T cell lineage requires signal transduction through the gammac, and once committed, their expansion requires signals relayed through the IL-7Ralpha.

Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice.

Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.

Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.

Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.

Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice.

C57BL/6 mice genetically deficient in interleukin 15 (IL-15(-/-) mice) were generated by gene targeting. IL-15(-/-) mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8(+) T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these populations in IL-15(-/-) mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15(-/-) mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15 in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15(-/-) mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15(-/-) mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid lineages. Moreover, the ability to rescue lymphoid defects in IL-15(-/-) mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.

Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers.

In mice deficient in either lymphotoxin-alpha (LT-alpha) or the type I tumor necrosis factor (TNF) receptor, but not the type II TNF receptor, germinal centers failed to develop in peripheral lymphoid organs. Germinal center formation was restored in LT-alpha-deficient mice by transplantation of normal bone marrow, indicating that the LT-alpha-expressing cells required to establish this lymphoid structure are derived from bone marrow.

An essential role for ectodomain shedding in mammalian development.

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Sequence homologies in the mouse protamine 1 and 2 genes.

To identify candidates for cis-acting sequences that regulate the stage and cell-specific expression of the two coordinately regulated protamine genes in the mouse, genomic clones were isolated and the nucleotide sequences of the 5' flanking regions and coding regions were compared. Unlike most histone genes and the multigene family of trout protamine genes which are intronless, each mouse protamine gene has a single, short intervening sequence. Although the coding regions do not share significant nucleotide homology, the 5' flanking regions contain several short homologous sequences that may be involved in gene regulation. An additional shared sequence is present in the 3' untranslated region surrounding the poly(A) addition signal in both genes.

Directed expression of an oncogene to Sertoli cells in transgenic mice using mullerian inhibiting substance regulatory sequences.

Mullerian inhibiting substance (MIS) is a glycoprotein hormone expressed by Sertoli cells that induces the regression of Mullerian ducts during development of the male reproductive tract. Transgenic mice carrying a fusion gene composed of human MIS transcriptional regulatory sequences linked to the SV40 T-antigen gene specifically develop testicular tumors composed of a cell type histologically resembling the Sertoli cell. The lack of pathology at other sites suggests tissue-restricted expression of the transgene. A cell line derived from one of the testicular tumors has been established that continues to express markers associated with Sertoli cells, such as transferrin, sulfated glycoprotein-2, and inhibin-beta B. The cell line does not express detectable levels of inhibin-alpha, MIS, or FSH receptor. However, the cells have retained forskolin responsiveness. As adult Sertoli cells cannot be propagated in vitro, the availability of an immortal cell line displaying features characteristic of normal Sertoli cells should aid in subsequent analyses of the biology of this cell type.

Defective T-cell receptor gamma gene rearrangement in interleukin-7 receptor knockout mice.

T-cell receptor (TCR) genes need to be rearranged by a site specific-VDJ recombinase before they are expressed. This process, initiated in CD44+25+ thymocytes, takes place during the early stage of T-cell differentiation in the thymus. Interleukin-7 receptor alpha chain knockout (IL-7R-/-) mice are severely deficient in B-lymphocytes and alpha beta T-cells and completely lack the gamma delta T-cell lineage. Thymocyte development is arrested at a very early stage (DN CD44+CD25-). Because this arrest is earlier than in mice with a block in VDJ recombination, we examined the rearrangement status of TCR genes in thymocytes from IL-7R-/- mice. The TCR beta locus showed a nearly normal pattern of VDJ rearrangements, consistent with the presence of alpha beta T-cells in these mice. However, TCR gamma locus rearrangement was absent or severely reduced for all the V gamma genes analyzed (V gamma 3, V gamma 4, V gamma 1.1, V gamma 1.2 and V gamma 2). In contrast, the delta locus showed little reduction in rearrangement. The defect in gamma rearrangements in IL-7R-/- thymocytes is not simply due to an absence of mature gamma delta T-cells, since TCR delta-/- mice, which also have only alpha beta T-cells, had normal levels of gamma and delta rearrangements. These findings indicate that one or both of the two known ligands of IL-7R, IL-7 and thymic stromal lymphopoietin (TSLP) serves as an extrinsic signal to specifically rearrange the TCR gamma locus.

Expression of mouse protamine 1 genes in transgenic mice.

Mouse protamine genes are expressed exclusively in spermatids. Mouse protamine 1 (mP1) transcriptional regulatory elements can target the expression of either marked mP1 transgenes or mP1 chimeric genes to spermatids in transgenic mice. Sequences between -40 and -465 bp relative to the transcription start site are required for expression in spermatids, whereas sequences 3' of the point of translation initiation are dispensable. mP1 transcriptional regulatory sequences were used to direct the expression of a toxic gene product to spermatids. The phenotypic consequences of toxin expression in spermatids are described.

Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.

The mechanisms that initiate liver regeneration after resection of liver tissue are not known. To determine whether cytokines are involved in the initiation of liver growth, we studied the regeneration of the liver after partial hepatectomy (PH) in mice lacking type I tumor necrosis factor receptor (TNFR-I). DNA synthesis after PH was severely impaired in these animals, and the expected increases in the binding of the NF-kappaB and STAT3 transcription factors shortly after PH failed to occur. Binding of AP-1 after PH was decreased in TNFR-I knockout mice compared with animals with the intact receptor whereas C/EBP binding was not modified. Injection of interleukin 6 in TNFR-I-deficient animals 30 min before PH corrected the defect in DNA synthesis and restored STAT3 and AP-1 binding to normal levels but had no effect on NF-kappaB binding in the regenerating liver. The results indicate that TNF, signaling through the TNFR-I, can initiate liver regeneration and acts by activating an interleukin 6-dependent pathway that involves the STAT3 transcription factor.

Roles of tumor necrosis factor receptor signaling during murine Escherichia coli pneumonia.

We hypothesized that tumor necrosis factor (TNF)-alpha signaling is essential to inflammation and host defense during Escherichia coli pneumonia. We tested this hypothesis by instilling E. coli into the lungs of wild-type (WT) mice and gene-targeted mice that lack both p55 and p75 receptors for TNF-alpha. The emigration of neutrophils 6 h after instillation of E. coli was not decreased, but rather was significantly increased (167% of WT), in TNF receptor (TNFR)-deficient mice. This increased neutrophil emigration did not result from peripheral blood neutrophilia or enhanced neutrophil sequestration, inasmuch as the numbers of neutrophils in the circulating blood and in the pulmonary capillaries did not differ between TNFR-deficient and WT mice. The accumulation of pulmonary edema fluid was not inhibited in TNFR-deficient compared with WT mice. Nuclear factor-kappaB (NF-kappaB) translocation in the lungs was not prevented in TNFR-deficient mice. Thus, signaling pathways independent of TNFRs can mediate the acute inflammatory response during E. coli pneumonia. However, despite this inflammatory response, bacterial clearance was impaired in TNFR-deficient mice (109 +/- 8% versus 51 +/- 14% of the original inoculum viable after 6 h in TNFR-deficient and WT mice, respectively). Increased neutrophil emigration during E. coli pneumonia in TNFR-deficient mice may thus result from an increased bacterial burden in the lungs. During acute E. coli pneumonia, the absence of TNFR signaling compromised bacterial killing, but did not prevent inflammation, as measured by the accumulation of edema fluid and neutrophils.

Accelerated atherosclerosis in mice lacking tumor necrosis factor receptor p55.

TNF-alpha (TNF) is produced primarily from macrophages and promotes numerous inflammatory reactions associated with atherosclerosis including the induction of vascular adhesion molecules and the recruitment and proliferation of monocyte/macrophages. There are two receptors known to elicit TNF responses, termed p55 and p75. Since p55 is thought to play the primary role in inflammatory processes, we postulated that the absence of p55 in mice would protect against atherosclerosis. In contrast, C57BL/6 mice lacking p55 had aortic sinus lesion sizes 2.3-fold larger than C57BL/6 wild type mice when fed an atherogenic diet (37,123 +/- 3485 microm2 versus 16, 688 +/- 2887 microm2, respectively, p < 0.0004). Plasma lipid levels were not different between strains. A 3-fold increase in the uptake and degradation of acetylated low density lipoprotein for p55-null as compared with wild type mice was demonstrated in cultured peritoneal macrophages. Immunohistochemical staining for scavenger receptor protein in the aortic sinus was more intense in lesions from the p55-null mice as compared with wild type controls. Our results support the concept that increased scavenger receptor activity contributes to excessive fatty streak formation. We conclude that TNF p55 receptors protect against atherosclerotic lesion development in the mouse.

Kinetics of enzymes subject to very strong product inhibition: analysis using simplified integrated rate equations and average velocities.

Enzymes which catalyze energetically unfavorable reactions in the physiological direction are likely to be strongly inhibited by the reaction products. (Some energetically favorable reactions may also display strong "product inhibition" when assayed in the reverse direction.) In some cases, the inhibition caused by an accumulating product is so potent that true initial velocities cannot be directly determined using conventional assay methods. Continuous removal of the inhibitory product may be mitigated against by the nature of the assay or the unavailability of the appropriate coupling enzyme. It can be shown that if (a) only one inhibitory product is allowed to accumulate and (b) the substrate concentrations remain essentially constant over the assay period (i.e. Kproduct less than or equal to 10(-2)Ksubstrate, so that the decreasing reaction rate stems only from progressive product inhibition), then plots of reciprocal average (apparent) velocity (i.e. 1/v = t/[P]) versus [P] are linear and extrapolate to 1/v0, the reciprocal of the initial uninhibited velocity at the fixed substrate concentrations. Intercept replots give the usual initial velocity reciprocal plot patterns and permit Vmax and the substrate Km's to be determined. Slope replots are diagnostic of the type of inhibition exerted by the accumulating product and permit the inhibition constants to be determined. If all the appropriate coupling enzymes are available, some kinetic mechanisms can be diagnosed using data derived from the reaction progress curves in the presence of one accumulating product at a time.

Spermatid-specific expression of protamine 1 in transgenic mice.

Protamines are abundant basic proteins involved in the condensation of sperm chromatin. In the mouse, protamine genes are transcribed postmeiotically in round spermatids. We have cloned and sequenced the mouse protamine 1 gene. Ten lines of transgenic mice harboring marked protamine 1 sequences were generated by microinjection of fertilized eggs. Transcription of the transgene is restricted to round spermatids and in several cases exceeds that of the endogenous gene. The cis-acting sequences required for tissue-specific protamine expression reside on a 2.4-kilobase restriction fragment. Prospects for using transgenic mice to address fundamental questions of male germ-cell development are discussed.

Antibody-induced shedding of CD44 from adherent cells is linked to the assembly of the cytoskeleton.

CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion receptor for the extracellular matrix glycosaminoglycan hyaluronan. CD44 participates in a variety of physiological and pathological processes through its role in cell adhesion. Under appropriate conditions, the ectodomain of CD44 is proteolytically removed from the cell surface. In this study we show that excessive CD44 shedding can be induced in mouse fibroblasts and monocytes upon exposure of these cells to a CD44-specific Ab immobilized on plastic, whereas treatment with phorbol ester induces significantly enhanced CD44 release from the monocytes only. CD44 shedding proceeds normally in fibroblasts and monocytes deficient in TNF-alpha converting enzyme (TACE), a sheddase involved in the processing of several substrates. Conversely, activation of the CD44 protease has no effect on the release of TNF-alpha from TACE-expressing cells, although the same metalloprotease inhibitor effectively blocks both TACE and the CD44 sheddase. Concomitant with anti-CD44 Ab- or phorbol ester-induced CD44 shedding, dramatic changes are observed in cell morphology and the structure of the actin cytoskeleton. Disruption of actin assembly with cytochalasin reduces CD44 shedding, but not the release of TNF-alpha. Moreover, pharmacological activation of Rho family GTPases Rac1 and Cdc42, which regulate actin filament assembly into distinct cytoskeletal structures, has a profound effect on CD44 release. We conclude that the CD44 sheddase and TACE are distinct enzymes, and that Ab- and phorbol ester-enhanced cleavage of CD44 is controlled in a cell type-dependent fashion by Rho GTPases through the cytoskeleton.

An interleukin-7 internet for intestinal intraepithelial T cell development: knockout of ligand or receptor reveal differences in the immunodeficient state.

Both interleukin-7 (IL-7) and IL-7 receptor (R) gene knockout (IL-7-/- and IL-7R-/-) mice were employed in order to directly investigate the importance of the IL-7 and IL-7R signaling pathway for the development of intestinal intraepithelial lymphocytes (IEL). Loss of the IL-7R-specific gene resulted in complete deficiency of the gamma delta T cell lineage with lack of V gamma 4- and V gamma 7-specific messages in the epithelium of the gastrointestinal (GI) tract in comparison to control mice of the same genetic background (approximately 40%). Disruption of the IL-7-specific gene resulted in marked, but not complete depletion of gamma delta T cells (2-3%) in IEL. Furthermore, mRNA for both V gamma 4 and V gamma 7 genes were detected in the gamma delta IEL subset of IL-7-/- mice. The subtle differences between IL-7-/- and IL-7R-/- mice suggest that although IL-7 controls most of the expansion and/or development of gamma delta IEL, another ligand binding to the IL-7R also plays a discernable role. Furthermore, alpha beta IEL developed more slowly in IL-7R-/- mice when compared with ligand knockouts; however, the frequency of IEL T cells subsequently increased with age and normal levels of CD3+ T cells expressing the alpha beta TCR were detected by 2 and 3 months of age in IL-7-/- and IL-7R-/- mice, respectively. The direct comparison of IL-7-/- and IL-7R-/- mice clearly supports the hypothesis that both IL-7 and another IL-7R binding molecule can influence the development of gamma delta T cells in the intestinal epithelium.

Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4.

HER4 is a member of the epidermal growth factor receptor family and has an essential function in heart and neural development. Identification of two HER4 isoforms, HER4 JM-a and JM-b, which differ in their extracellular juxtamembrane region and in their susceptibility to cleavage after phorbol ester stimulation, showed that the juxtamembrane region of the receptor is critical for proteolysis. We now demonstrate that phorbol ester and pervanadate are effective stimuli for HER4 JM-a processing and that the HER4 JM-b isoform does not undergo cleavage in response to any of the stimuli studied. We also show that HER4 JM-a is not cleaved in cells lacking the metalloprotease tumor necrosis factor-alpha-converting enzyme (TACE) and that reexpression of TACE in these cells restores constitutive and regulated processing of HER4 JM-a. Moreover, we show that the sequence specific to the HER4 JM-a juxtamembrane region is sufficient to confer susceptibility to phorbol 12-myristate 13-acetate-induced cleavage of the HER2 receptor. In conclusion, we provide evidence that TACE is essential for the regulated shedding of the HER4 JM-a receptor.

TNF receptor-deficient mice reveal striking differences between several models of thymocyte negative selection.

Central tolerance depends upon Ag-mediated cell death in developing thymocytes. However, the mechanism of induced death is poorly understood. Among the known death-inducing proteins, TNF was previously found to be constitutively expressed in the thymus. The role of TNF in thymocyte negative selection was therefore investigated using TNF receptor (TNFR)-deficient mice containing a TCR transgene. TNFR-deficient mice displayed aberrant negative selection in two models: an in vitro system in which APC are cultured with thymocytes, and a popular in vivo system in which mice are treated with anti-CD3 Abs. In contrast, TNFR-deficient mice displayed normal thymocyte deletion in two Ag-induced in vivo models of negative selection. Current models of negative selection and the role of TNFR family members in this process are discussed in light of these results.