Issue published December 15, 2000

X -linked autoimmunity–allergic disregulation syndrome (XLAAD) is an X-linked recessive immunological disorder characterized by multisystem autoimmunity, particularly early-onset type 1 diabetes mellitus, associated with manifestations of severe atopy including eczema, food allergy, and eosinophilic inflammation. Consistent with the allergic phenotype, analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. Using a positional-candidate approach, we have identified in both kindreds mutations in JM2, a gene on Xp11.23 that encodes a fork head domain–containing protein. One point mutation at a splice junction site results in transcripts that encode a truncated protein lacking the fork head homology domain. The other mutation involves an in-frame, 3-bp deletion that is predicted to impair the function of a leucine zipper dimerization domain. Our results point to a critical role for JM2 in self tolerance and Th cell differentiation.This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R75–R81 (2000).

T he KvLQT1 gene encodes a voltage-gated potassium channel. Mutations in KvLQT1 underlie the dominantly transmitted Ward-Romano long QT syndrome, which causes cardiac arrhythmia, and the recessively transmitted Jervell and Lange-Nielsen syndrome, which causes both cardiac arrhythmia and congenital deafness. KvLQT1 is also disrupted by balanced germline chromosomal rearrangements in patients with Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth and cancer. Because of the diverse human disorders and organ systems affected by this gene, we developed an animal model by inactivating the murine Kvlqt1. No electrocardiographic abnormalities were observed. However, homozygous mice exhibited complete deafness, as well as circular movement and repetitive falling, suggesting imbalance. Histochemical study revealed severe anatomic disruption of the cochlear and vestibular end organs, suggesting that Kvlqt1 is essential for normal development of the inner ear. Surprisingly, homozygous mice also displayed threefold enlargement by weight of the stomach resulting from mucous neck cell hyperplasia. Finally, there were no features of BWS, suggesting that Kvlqt1 is not responsible for BWS.

C D18-deficient mice (CD18^–/– mice) have a severe leukocyte recruitment defect in some organs, and no detectable defect in other models. Mice lacking E-selectin (CD62E^–/– mice) have either no defect or a mild defect of neutrophil infiltration, depending on the model. CD18^–/–CD62E^–/–, but not CD18^–/–CD62P^–/–, mice generated by crossbreeding failed to thrive, reaching a maximum body weight of 10–15 grams. To explore the mechanisms underlying reduced viability, we investigated lethally irradiated CD62E^–/– mice that were reconstituted with CD18^–/– bone marrow. These mice, but not single-mutant controls, showed tenfold-increased rolling velocities in a TNF-α–induced model of inflammation. Leukocyte adhesion efficiency in CD18^–/–CD62E^–/– mice was reduced by 95%, and hematopoiesis was drastically altered, including severe bone marrow and blood neutrophilia and elevated G-CSF and GM-CSF levels. The greatly reduced viability of CD18^–/–CD62E^–/– mice appears to result from an inability to mount an adequate inflammatory response. Our data show that cooperation between E-selectin and CD18 integrins is necessary for neutrophil recruitment and that alternative adhesion pathways cannot compensate for the loss of these molecules.

P emphigus is an autoimmune disease of skin adhesion associated with autoantibodies against a number of keratinocyte antigens, such as the adhesion molecules desmoglein (Dsg) 1 and 3 and acetylcholine receptors. The notion that anti-Dsg antibodies alone are responsible for blisters in patients with pemphigus vulgaris (PV) stems from the ability of rDsg1 and rDsg3 to absorb antibodies that cause PV-like skin blisters in neonatal mice. Here, we demonstrate that PV IgGs eluted from rDsg1-Ig-His and rDsg3-Ig-His show similar antigenic profiles, including the 38-, 43-, 115-, and 190-kDa keratinocyte proteins and a non–Dsg 3 130-kDa polypeptide present in keratinocytes from Dsg 3 knockout mouse. We injected into Dsg 3–lacking mice the PV IgGs that did not cross-react with the 160-kDa Dsg 1 or its 45-kDa immunoreactive fragment and that showed no reactivity with recombinant Dsg 1. We used both the Dsg3^null mice with a targeted mutation of the Dsg3 gene and the “balding” Dsg3^bal/Dsg3^bal mice that carry a spontaneous null mutation in Dsg3. These PV IgGs caused gross skin blisters with PV-like suprabasal acantholysis and stained perilesional epidermis in a fishnet-like pattern, indicating that the PV phenotype can be induced without anti–Dsg 3 antibody. The anti–Dsg 1 antibody also was not required, as its presence in PV IgG does not alter the PV-like phenotype in skin organ cultures and because pemphigus foliaceus IgGs produce a distinct phenotype in Dsg3^null mice. Therefore, mucocutaneous lesions in PV patients could be caused by non-Dsg antibodies.

W hile TNF-α is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-α alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NF-κB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-α and RANKL markedly potentiate NF-κB and stress-activated protein kinase/c-Jun NH₂-terminal kinase activity, two signaling pathways essential for osteoclastogenesis. In vivo administration of TNF-α prompts robust osteoclast formation in chimeric animals in which β-galactosidase positive, TNF-responsive macrophages develop within a TNF-nonresponsive stromal environment. Thus, while TNF-α alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages within a stromal environment that expresses permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-α to promote osteoclastogenesis, TNF-α appears to be a more convenient target in arresting inflammatory osteolysis.

E limination of amyloid-β peptide (Aβ) from the brain is poorly understood. After intracerebral microinjections in young mice, ¹²⁵I-Aβ_1-40 was rapidly removed from the brain (t_1/2 ≤ 25 minutes), mainly by vascular transport across the blood-brain barrier (BBB). The efflux transport system for Aβ_1-40 at the BBB was half saturated at 15.3 nM, and the maximal transport capacity was reached between 70 nM and 100 nM. Aβ_1-40 clearance was substantially inhibited by the receptor-associated protein, and by antibodies against LDL receptor–related protein-1 (LRP-1) and α₂-macroglobulin (α₂M). As compared to adult wild-type mice, clearance was significantly reduced in young and old apolipoprotein E (apoE) knockout mice, and in old wild-type mice. There was no evidence that Aβ was metabolized in brain interstitial fluid and degraded to smaller peptide fragments and amino acids before its transport across the BBB into the circulation. LRP-1, although abundant in brain microvessels in young mice, was downregulated in older animals, and this downregulation correlated with regional Aβ accumulation in brains of Alzheimer’s disease (AD) patients. We conclude that the BBB removes Aβ from the brain largely via age-dependent, LRP-1–mediated transport that is influenced by α₂M and/or apoE, and may be impaired in AD.

A po-E–deficient apo-B100–only mice (Apoe^–/–Apob^100/100) and LDL receptor–deficient apo-B100–only mice (Ldlr^–/–Apob^100/100) have similar total plasma cholesterol levels, but nearly all of the plasma cholesterol in the former animals is packaged in VLDL particles, whereas, in the latter, plasma cholesterol is found in smaller LDL particles. We compared the apo-B100–containing lipoprotein populations in these mice to determine their relation to susceptibility to atherosclerosis. The median size of the apo-B100–containing lipoprotein particles in Apoe^–/–Apob^100/100 plasma was 53.4 nm versus only 22.1 nm in Ldlr^–/–Apob^100/100 plasma. The plasma levels of apo-B100 were three- to fourfold higher in Ldlr^–/–Apob^100/100 mice than in Apoe^–/–Apob^100/100 mice. After 40 weeks on a chow diet, the Ldlr^–/–Apob^100/100 mice had more extensive atherosclerotic lesions than Apoe^–/–Apob^100/100 mice. The aortic DNA synthesis rate and the aortic free and esterified cholesterol contents were also higher in the Ldlr^–/–Apob^100/100 mice. These findings challenge the notion that all non-HDL lipoproteins are equally atherogenic and suggest that at a given cholesterol level, large numbers of small apo-B100–containing lipoproteins are more atherogenic than lower numbers of large apo-B100–containing lipoproteins.

U sing a rat model of ischemia/reperfusion injury, we demonstrate here that HGF is cardioprotective due to its antiapoptotic effect on cardiomyocytes. Following transient myocardial ischemia and reperfusion, c-Met/HGF receptor expression rapidly increased in the ischemic myocardium, an event accompanied by a dramatic increase in plasma HGF levels in the infarcted rats. When endogenous HGF was neutralized with a specific antibody, the number of myocyte cell deaths increased markedly, the infarct area expanded, and the mortality increased to 50%, as compared with a control group in which there was no mortality. Plasma from the myocardial infarcted rats had cardioprotective effects on primary cultured cardiomyocytes, but these effects were significantly diminished by neutralizing HGF. In contrast, recombinant HGF administration reduced the size of infarct area and improved cardiac function by suppressing apoptosis in cardiomyocytes. HGF rapidly augmented Bcl-xL expression in injured cardiomyocytes both in vitro and in vivo. As apoptosis of cardiomyocytes is one of the major contributors to the pathogenesis in subjects with ischemia/reperfusion injury, prevention of apoptosis may prove to be a reasonable therapeutic strategy. Supplements of HGF, an endogenous cardioprotective factor, may be found clinically suitable in treating subjects with myocardial infarction.

T he endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H₂O₂) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H₂O₂ to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K⁺ channel opener levcromakalim. Exogenous H₂O₂ elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H₂O₂ upon stimulation by ACh and that the H₂O₂ production was markedly reduced in eNOS-KO mice. These results indicate that H₂O₂ is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.

P rotein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI^–/– mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.

R otavirus (RV), which replicates exclusively in cells of the small intestine, is the most important cause of severe diarrhea in young children worldwide. Using a mouse model, we show that expression of the intestinal homing integrin α₄β₇ is not essential for CD8⁺ T cells to migrate to the intestine or provide immunity to RV. Mice deficient in β7 expression (β7^–/–) and unable to express α₄β₇ integrin were found to clear RV as quickly as wild-type (wt) animals. Depletion of CD8⁺ T cells in β7^–/– animals prolonged viral shedding, and transfer of immune β7^–/– CD8⁺ T cells into chronically infected Rag-2–deficient mice resolved RV infection as efficiently as wt CD8⁺ T cells. Paradoxically, α₄β₇^hi memory CD8⁺ T cells purified from wt mice that had been orally immunized cleared RV more efficiently than α₄β₇^low CD8⁺ T cells. We explained this apparent contradiction by demonstrating that expression of α₄β₇ on effector CD8⁺ T cells depends upon the site of initial antigen exposure: oral immunization generates RV-specific CD8⁺ T cells primarily of an α₄β₇^hi phenotype, but subcutaneous immunization yields both α₄β₇^hi and α₄β₇^low immune CD8⁺ T cells with anti-RV effector capabilities. Thus, α₄β₇ facilitates normal intestinal immune trafficking to the gut, but it is not required for effective CD8⁺ T cell immunity.

Y oung adult males who cannot produce or respond to estrogen (E) are osteopenic, suggesting that E may regulate bone turnover in men, as well as in women. Both bioavailable E and testosterone (T) decrease substantially in aging men, but it is unclear which deficiency is the more important factor contributing to the increased bone resorption and impaired bone formation that leads to their bone loss. Thus, we addressed this issue directly by eliminating endogenous T and E production in 59 elderly men (mean age 68 years), studying them first under conditions of physiologic T and E replacement and then assessing the impact on bone turnover of withdrawing both T and E, withdrawing only T, or only E, or continuing both. Bone resorption markers increased significantly in the absence of both hormones and were unchanged in men receiving both hormones. By two-factor ANOVA, E played the major role in preventing the increase in the bone resorption markers, whereas T had no significant effect. By contrast, serum osteocalcin, a bone formation marker, decreased in the absence of both hormones, and both E and T maintained osteocalcin levels. We conclude that in aging men, E is the dominant sex steroid regulating bone resorption, whereas both E and T are important in maintaining bone formation.

A ctivation of peripheral blood T cells results in a rapid and substantial rise in translation rates and proliferation, but proliferation in response to mitogen stimulation is impaired in systemic lupus erythematosus (SLE). We have investigated translation rates and initiation factor activities in T cells from SLE patients in response to activating signals. Activation by PMA plus ionomycin strongly increased protein synthesis in control T cells but not in T cells from SLE patients. The rate of protein synthesis is known to be strongly dependent on the activity of two eukaryotic translation initiation factors, eIF4E and eIF2α. We show that following stimulation, eIF4E expression and phosphorylation increased equivalently in control and SLE T cells. Expression of eIF4E interacting proteins — eIF4G, an inducer, and 4E-BP1 and 4E-BP2, two specific repressors of eIF4E function — and the phosphorylation level of 4E-BP1, were all identical in control and SLE T cells. In contrast, the protein kinase PKR, which is responsible for the phosphorylation and consequent inhibition of eIF2α activity, was specifically overexpressed in activated SLE T cells, correlating with an increase in eIF2α phosphorylation. Therefore, high expression of PKR and subsequent eIF2α phosphorylation is likely responsible, at least in part, for impaired translational and proliferative responses to mitogens in T cells from SLE patients.