Issue published August 1, 1999

W e recently identified a new gene, klotho, which is involved in the suppression of multiple aging phenotypes. The mouse homozygous for a disruption of the klotho locus (kl/kl) exhibited multiple pathological conditions resembling human aging. Histomorphometric analysis revealed low-turnover osteopenia in kl/kl mice. The decrease in bone formation exceeded that of bone resorption, resulting in a net bone loss. The number of osteoblast progenitors determined by ex vivo bone marrow cultures was reduced in kl/kl mice. In addition, cultured osteoblastic cells derived from kl/kl mice showed lower alkaline phosphatase activity and matrix nodule formation than those from wild-type mice. Osteoclastogenesis in the coculture of marrow cells and osteoblastic cells was decreased only when marrow cells originated from kl/kl mice independently of the origin of osteoblastic cells. We also found that the expression of osteoprotegerin, an osteoclastogenesis inhibitor, was significantly upregulated in kl/kl mice. We conclude that a defect in the klotho gene expression causes the independent impairment of both osteoblast and osteoclast differentiation, leading to low-turnover osteopenia. Because this state represents a characteristic feature of senile osteoporosis in humans, kl/kl mice can be regarded as a useful model for investigating cellular and molecular mechanisms of age-related bone loss.

I schemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH]_o). Apoptosis did not occur when [pH]_o was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis–mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25–30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53.J. Clin. Invest. 104:239–252 (1999).

G alanin is widely distributed in enteric nerve terminals lining the human gastrointestinal (GI) tract. We have shown previously that galanin-1 receptors (Gal1-R) are expressed by epithelial cells lining the human GI tract, and upon activation cause Cl^– secretion. Because expression of this receptor is transcriptionally regulated by nuclear factor-κB (NF-κB), which is activated by enteric pathogens as a part of the host epithelial response to infection, we investigated whether such bacterial pathogens could directly increase Gal1-R expression in the T84-cell model system. Pathogenic Escherichia coli, but not nonpathogenic E. coli, activate a p50/p65 NF-κB complex that binds to oligonucleotides corresponding to a recognition site located within the 5′ flanking region of the human GAL1R gene. Pathogenic E. coli, but not normal commensal organisms, increase Gal1-R mRNA synthesis and [¹²⁵I]galanin binding sites. Whereas galanin increases short-circuit current (Isc) approximately 5-fold in uninfected T84 cells, exposure to pathogenic, but not nonpathogenic, E. coli results in galanin increasing Isc approximately 20-fold. To confirm the validity of these in vitro observations, we also studied C57BL/6J mice infected with enterohemorrhagic E. coli (EHEC) by gavage. Infection caused a progressive increase in both NF-κB activation and Gal1-R expression, with maximal levels of both observed 3 days after gavage. Ussing chamber studies revealed that colons infected with EHEC, but not those exposed to normal colonic flora, markedly increased Isc in response to galanin. These data indicate that pathogen-induced increases in Gal1-R expression by epithelial cells lining the colon may represent a novel unifying pathway responsible for at least a portion of the excessive fluid secretion observed during infectious diarrhea.

W e have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.

E xpression of innate immune response proteins, including IL-1β, TNF, and the cytokine-inducible isoform of nitric oxide synthase (iNOS), have been documented in the hearts of humans and experimental animals with heart failure regardless of etiology, although the proximal events leading to their expression are unknown. Noting that expression of a human homologue of Drosophila Toll, a proximal innate immunity transmembrane signaling protein in the fly, now termed human Toll-like receptor 4 (hTLR4), appeared to be relatively high in the heart, we examined TLR4 mRNA and protein abundance in isolated cellular constituents of cardiac muscle and in normal and abnormal murine, rat, and human myocardium. TLR4 expression levels in cardiac myocytes and in coronary microvascular endothelial cells could be enhanced by either LPS or IL-1β, an effect inhibited by the oxygen radical scavenger PDTC. Transfection of a constitutively active TLR4 construct, CD4/hTLR4, resulted in activation of a nuclear factor-κB reporter construct, but not of an AP-1 or an iNOS reporter construct, in cardiac myocytes. In normal murine, rat, and human myocardium, TLR4 expression was diffuse, and presumably cytoplasmic, in cardiac myocytes. However, in remodeling murine myocardium remote from sites of ischemic injury and in heart tissue from patients with idiopathic dilated cardiomyopathy, focal areas of intense TLR4 staining were observed in juxtaposed regions of 2 or more adjacent myocytes; this staining was not observed in control myocardium. Increased expression and signaling by TLR4, and perhaps other Toll homologues, may contribute to the activation of innate immunity in injured myocardium.J. Clin. Invest. 104:271–280 (1999).

G alα1,3Gal–reactive (Gal-reactive) antibodies are a major impediment to pig-to-human xenotransplantation. We investigated the potential to induce tolerance of anti-Gal–producing cells and prevent rejection of vascularized grafts in the combination of α1,3-galactosyltransferase wild-type (GalT^+/+) and deficient (GalT^–/–) mice. Allogeneic (H-2 mismatched) GalT^+/+ bone marrow transplantation (BMT) to GalT^–/– mice conditioned with a nonmyeloablative regimen, consisting of depleting CD4 and CD8 mAb’s and 3 Gy whole-body irradiation and 7 Gy thymic irradiation, led to lasting multilineage H-2^bxd GalT^+/+ + H-2^d GalT^–/– mixed chimerism. Induction of mixed chimerism was associated with a rapid reduction of serum anti-Gal naturally occurring antibody levels. Anti-Gal–producing cells were undetectable by 2 weeks after BMT, suggesting that anti-Gal–producing cells preexisting at the time of BMT are rapidly tolerized. Even after immunization with Gal-bearing xenogeneic cells, mixed chimeras were devoid of anti-Gal–producing cells and permanently accepted donor-type GalT^+/+ heart grafts (>150 days), whereas non-BMT control animals rejected these hearts within 1–7 days. B cells bearing receptors for Gal were completely absent from the spleens of mixed chimeras, suggesting that clonal deletion and/or receptor editing may maintain B-cell tolerance to Gal. These findings demonstrate the principle that induction of mixed hematopoietic chimerism with a potentially relevant nonmyeloablative regimen can simultaneously lead to tolerance among both T cells and Gal-reactive B cells, thus preventing vascularized xenograft rejection.

T hyroid hormone receptors (TRs) modulate various physiological functions in many organ systems. The TRα and TRβ isoforms are products of 2 distinct genes, and the β1 and β2 isoforms are splice variants of the same gene. Whereas TRα1 and TRβ1 are widely expressed, expression of the TRβ2 isoform is mainly limited to the pituitary, triiodothyronine-responsive TRH neurons, the developing inner ear, and the retina. Mice with targeted disruption of the entire TRβ locus (TRβ-null) exhibit elevated thyroid hormone levels as a result of abnormal central regulation of thyrotropin, and also develop profound hearing loss. To clarify the contribution of the TRβ2 isoform to the function of the endocrine and auditory systems in vivo, we have generated mice with targeted disruption of the TRβ2 isoform. TRβ2-null mice have preserved expression of the TRα and TRβ1 isoforms. They develop a similar degree of central resistance to thyroid hormone as TRβ-null mice, indicating the important role of TRβ2 in the regulation of the hypothalamic-pituitary-thyroid axis. Growth hormone gene expression is marginally reduced. In contrast, TRβ2-null mice exhibit no evidence of hearing impairment, indicating that TRβ1 and TRβ2 subserve divergent roles in the regulation of auditory function.

E arly-phase reactions (EPRs) and late-phase reactions (LPRs) are characteristic features of bronchial asthma, although the pathogenetic mechanisms responsible for each of the responses are not fully defined. A murine model of EPRs and LPRs was developed to investigate the role of IL-5 and eosinophils in development of both responses. After initial intraperitoneal sensitization and airway challenge to ovalbumin (OVA), mice were provoked by additional exposure to OVA. An EPR, characterized by a transient increase in airway responsiveness, was observed 5–30 minutes after antigen provocation. This response was followed by an LPR that reached its maximum at 6 hours after challenge and was characterized by increased airway responsiveness and significant lung eosinophilia. The EPR was blocked by cromoglycate and albuterol, whereas the LPR was abolished by cromoglycate and hydrocortisone. Before provocation with allergen, administration of anti–IL-5 antibody prevented the influx of eosinophils into the lung tissue and abolished the LPR but not EPR. These results suggest that IL-5 and eosinophils are essential for development of the LPR, but not EPR, in this model.

P olymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B₄ receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A₄ was produced in ischemic lungs and elevated by reperfusion. Because LXA₄ and aspirin-triggered 15-epimeric LXA₄ (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA₄ and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

W e recently showed that IL-11 prevents lethal graft-versus-host disease (GVHD) in a murine bone marrow transplantation (BMT) model of GVHD directed against MHC and minor antigens. In this study, we have investigated whether IL-11 can maintain a graft-versus-leukemia (GVL) effect. Lethally irradiated B6D2F1 mice were transplanted with either T cell–depleted (TCD) bone marrow (BM) alone or with BM and splenic T cells from allogeneic B6 donors. Animals also received host-type P815 mastocytoma cells at the time of BMT. Recipients were injected subcutaneously with recombinant human IL-11 or control diluent twice daily, from 2 days before BMT to 7 days after BMT. TCD recipients all died from leukemia by day 23. All control- and IL-11–treated allogeneic animals effectively rejected their leukemia, but IL-11 also reduced GVHD-related mortality. Examination of the cellular mechanisms of GVL and GVHD in this system showed that IL-11 selectively inhibited CD4-mediated GVHD, while retaining both CD4- and CD8-mediated GVL. In addition, IL-11 treatment did not affect cytolytic effector functions of T cells after BMT either in vivo or in vitro. Studies with perforin-deficient donor T cells demonstrated that the GVL effect was perforin dependent. These data demonstrated that IL-11 can significantly reduce CD4-dependent GVHD without impairing cytolytic function or subsequent GVL activity of CD8⁺ T cells. Brief treatment with IL-11 shortly after BMT may therefore represent a novel strategy for separating GVHD and GVL.

S ecretion of inflammatory products from neutrophils can be induced by a combination of signals from ligated integrins and receptors for soluble, physiological agonists such as TNF. Here we identify pyk2 in primary human neutrophils; localize it to focal adhesions and podosomes; and demonstrate its tyrosine phosphorylation, activation, and association with paxillin during stimulation of adherent cells by TNF. Tyrphostin A9 emerged as the most potent and selective of 51 tyrosine kinase inhibitors tested against the TNF-induced respiratory burst. Tyrphostin A9 inhibited TNF-induced tyrosine phosphorylation of pyk2 without blocking the cells’ bactericidal activity. Wortmannin, an inhibitor of phosphatidylinositol-3-kinase, potently blocked the TNF-induced respiratory burst and selectively inhibited tyrosine phosphorylation of pyk2. Thus, pyk2 appears to play an essential role in the ability of neutrophils to integrate signals from β₂ integrins and TNF receptors.

T raditional enzyme kinetics provide a poor explanation for the increased risk of lung injury in α₁-antitrypsin (AAT) deficiency. Millimolar concentrations of leukocyte elastase, when released from single azurophil granules of activated neutrophils, lead to evanescent quantum bursts of proteolytic activity before catalysis is quenched by pericellular inhibitors. Herein, we tested the possibility that quantum proteolytic events are abnormal in AAT deficiency. We incubated neutrophils on opsonized fluoresceinated fibronectin in serum from individuals with various AAT phenotypes, and then measured and modeled quantum proteolytic events. The mean areas of the events in serum from heterozygous individuals (Pi MZ and Pi SZ) were slightly, but significantly, larger than those in serum from normal patients (Pi M). In marked contrast, mean areas of events in serum from AAT-deficient individuals were 10-fold larger than those in serum from normal patients. Diffusion modeling predicted that local elastase concentrations exceed AAT concentrations for less than 20 milliseconds and for more than 80 milliseconds in Pi M and Pi Z individuals, respectively. Thus, quantum proteolytic events are abnormally large and prolonged in AAT deficiency, leading directly to an increased risk of tissue injury in the immediate vicinity of activated neutrophils. These results have potentially important implications for the pathogenesis and prevention of lung disease in AAT deficiency.

W e tested 154 peptides spanning the entire length of core histones of nucleosomes for the ability to stimulate an anti-DNA autoantibody–inducing T helper (Th) clone, as well as CD4⁺ T-cell lines and T cells, in fresh PBMCs from 23 patients with lupus erythematosus. In contrast to normal T cells, lupus T cells responded strongly to certain histone peptides, irrespective of the patient’s disease status. Nucleosomal peptides in histone regions H2B_10–33, H4_16–39 (and overlapping H4_14–28), H4_71–94, and H3_91–105 (and overlapping H3_100–114) were recurrently recognized by CD4 T cells from the patients with lupus. Remarkably, these same peptides overlap with major epitopes for the Th cells that induce anti-DNA autoantibodies and nephritis in lupus-prone mice. We localized 2 other recurrent epitopes for human lupus T cells in H2A_34–48 and H4_49–63. All the T-cell autoepitopes have multiple HLA-DR binding motifs, and the epitopes are located in histone regions recognized by lupus autoantibodies, suggesting a basis for their immunodominance. Native nucleosomes and their peptides H4_16–39, H4_71–94, and H3_91–105 induced a stronger IFN-γ response, whereas others, particularly, H2A_34–48, favored an IL-10– and/or IL-4–positive T-cell response. The major autoepitopes may reveal the mechanism of autoimmune T-cell expansion and lead to antigen-specific therapy of human lupus.

R ecent studies have identified several polymorphisms in the human insulin receptor substrate-1 (IRS-1) gene. The most prevalent IRS-1 variant, a Gly→Arg change at the codon 972, has been reported to be increased in prevalence among patients with type 2 diabetes. Carriers of the Arg⁹⁷² substitution are characterized by lower fasting insulin and C-peptide levels compared with non-carriers, suggesting that the Arg⁹⁷² IRS-1 variant may contribute to impairment of insulin secretion. In this study, we stably overexpressed both wild-type IRS-1 (RIN-WT) and Arg⁹⁷² IRS-1 variant (RIN-Arg⁹⁷²) in RIN β cells to investigate directly whether the polymorphism in codon 972 of IRS-1 impairs insulin secretion. The Arg⁹⁷² IRS-1 variant did not affect expression or function of endogenous IRS-2. RIN-WT showed a marked increase in both glucose- and insulin-stimulated tyrosine phosphorylation of IRS-1 compared with control RIN cells. The Arg⁹⁷² IRS-1 variant did not alter the extent of either glucose- or insulin-stimulated tyrosine phosphorylation of recombinant IRS-1. However, RIN-Arg⁹⁷² showed a significant decrease in binding of the p85 subunit of phosphatidylinositol-3-kinase (PI 3-kinase) with IRS-1, compared with RIN-WT. Compared with control RIN cells, insulin content was reduced to the same extent in RIN-WT or RIN-Arg⁹⁷² at both the protein and mRNA levels. Both glucose- and sulfonylurea-induced insulin secretion was increased in RIN-WT compared with control RIN cells. By contrast, RIN cells expressing Arg⁹⁷² IRS-1 exhibited a marked decrease in both glucose- and sulfonylurea-stimulated insulin secretion compared with RIN-WT. These data suggest that the insulin signaling pathway involving the IRS-1/PI 3-kinase may play an important role in the insulin secretory process in pancreatic β cells. More importantly, the results suggest that the common Arg⁹⁷² IRS-1 polymorphism may impair glucose-stimulated insulin secretion, thus contributing to the relative insulin deficiency observed in carriers of this variant.