Issue published October 15, 2002

H aving recently described the injurious role of caspase-1–mediated production of the proinflammatory cytokine IL-18 in ischemic acute renal failure (ARF), we report here on the effect of the newly developed caspase inhibitor Quinoline-Val-Asp(Ome)-CH₂-OPH (OPH-001) on caspase-1, IL-18, neutrophil infiltration, and renal function in ischemic ARF. C57BL/6 mice with ischemic ARF treated with OPH-001 had a marked (100%) reduction in blood urea nitrogen (BUN) and serum creatinine and a highly significant reduction in morphological acute tubular necrosis (ATN) score compared with vehicle-treated mice. OPH-001 significantly reduced the increase in caspase-1 activity and IL-18 and prevented neutrophil infiltration in the kidney during ischemic ARF. To evaluate whether this lack of neutrophil infiltration was contributing to the protection against ischemic ARF, a model of neutrophil depletion was developed. Neutrophil-depleted mice had a small (18%) reduction in serum creatinine during ischemic ARF but no reduction in ATN score despite a lack of neutrophil infiltration in the kidney. Remarkably, caspase-1 activity and IL-18 were significantly increased in the kidney in neutrophil-depleted mice with ARF. In addition, IL-18 antiserum–treated neutrophil-depleted mice with ischemic ARF had a significant (75%) reduction in serum creatinine and a significant reduction in ATN score compared with vehicle-treated neutrophil-depleted mice. These results suggest a novel neutrophil-independent mechanism of IL-18–mediated ischemic ARF.

T he wide range of phenotypic abnormalities seen in the leptin-deficient ob/ob mouse and their reversibility by leptin administration provide compelling evidence for the existence of multiple physiological functions of this hormone in rodents. In contrast, information regarding the roles of this hormone in humans is limited. Three morbidly obese children, who were congenitally deficient in leptin, were treated with daily subcutaneous injections of recombinant human leptin for up to 4 years with sustained, beneficial effects on appetite, fat mass, hyperinsulinemia, and hyperlipidemia. Leptin therapy resulted in a rapid and sustained increase in plasma thyroid hormone levels and, through its age-dependent effects on gonadotropin secretion, facilitated appropriately timed pubertal development. Leptin deficiency was associated with reduced numbers of circulating CD4⁺ T cells and impaired T cell proliferation and cytokine release, all of which were reversed by recombinant human leptin administration. The subcutaneous administration of recombinant human leptin has major and sustained beneficial effects on the multiple phenotypic abnormalities associated with congenital human leptin deficiency.

I n this report, we demonstrate that herpes simplex virus (HSV) infection of the cornea results in the upregulation of the matrix-degrading metalloproteinase enzyme MMP-9. This enzyme was shown to contribute to the neovascularization process that occurs in the corneal stroma in response to HSV infection. The likely source of MMP-9, at least initially after infection, was neutrophils that were signaled to invade the cornea soon after infection. Corneal infiltrating neutrophils were shown to express MMP-9, and preventing the neutrophil response with specific mAb diminished MMP-9 expression as well as the extent of angiogenesis. Further supporting a role for MMP-9 in HSV-induced corneal angiogenesis was the observation that inhibition of MMP-9 with the specific inhibitor TIMP-1 resulted in reduced angiogenesis. In addition, angiogenesis was diminished in ocularly infected MMP-9 knockout mice. Our results demonstrate that MMP-9 is involved in angiogenesis caused by HSV. Since angiogenesis appears to represent a vital step in the pathogenesis of herpetic stromal keratitis, these results indicate that targeting MMP-9 for inhibition should prove useful for the therapy of herpetic stromal keratitis.

T he recruitment of antigen-specific T lymphocytes to the intestinal mucosa is central to the development of an effective mucosal immune response, yet the mechanism by which this process occurs remains to be fully defined. Here we show that the CC chemokine receptor 9 (CCR9) is selectively and functionally expressed on murine α_Eβ₇⁺ naive CD8αβ⁺ lymphocytes and a subset of recently activated CD69⁺ CD8αβ⁺ lymphocytes. Using a T cell receptor transgenic transfer model, we demonstrate that CCR9 expression is functionally maintained on CD8αβ⁺ lymphocytes following activation in mesenteric lymph nodes but rapidly downregulated on CD8αβ⁺ lymphocytes activated in peripheral lymph nodes. These recently activated CCR9⁺ CD8αβ⁺ lymphocytes selectively localized to the small-intestinal mucosa, and in vivo neutralization of the CCR9 ligand, CCL25, reduced the ability of these cells to populate the small-intestinal epithelium. Together these results demonstrate an important role for chemokines in the localization of T lymphocytes to the small-intestinal mucosa and suggest that targeting CCL25 and/or CCR9 may provide a means to selectively modulate small-intestinal immune responses.

T he properties of a proapoptotic 1,4-benzodiazepine, Bz-423, identified through combinatorial chemistry and phenotype screening are described. Bz-423 rapidly generated superoxide (O₂^–) in transformed Ramos B cells. This O₂^– response originated from mitochondria prior to mitochondrial transmembrane gradient collapse and opening of the permeability transition pore. Bz-423–induced O₂^– functioned as an upstream signal that initiated an apoptotic program characterized by cytochrome c release, mitochondrial depolarization, and caspase activation. Pretreatment of cells with agents that either block the formation of Bz-423–induced O₂^– or scavenge free radicals attenuated the death cascade, which demonstrated that cell killing by Bz-423 depends on O₂^–. Parallels between Ramos cells and germinal center B cells prompted experiments to determine whether Bz-423 had therapeutic activity in vivo. This possibility was tested using the (NZB × NZW)F₁ murine model of lupus, in which the pathologically enhanced survival and expansion of germinal center B cells mediate disease. Administration of Bz-423 for 12 weeks specifically controlled germinal center hyperplasia and reduced the histological evidence of glomerulonephritis. Collectively, these studies define a new structure-function relationship for benzodiazepines and point to a new target and mechanism that could be of value for developing improved drugs to manage systemic lupus erythematosus and related disorders.

L ymphatic tissues (LTs) are structurally organized to promote interaction between antigens, chemokines, growth factors, and lymphocytes to generate an immunologic response and maintain normal-sized populations of CD4⁺ and CD8⁺ T cells. Inflammation and tissue remodeling that accompany local innate and adaptive immune responses to HIV-1 replication cause damage to the LT architecture. As a result, normal populations of CD4⁺ and CD8⁺ T cells cannot be supported and antigen-lymphocyte interactions are impaired. This conclusion is supported herein following LT sampling before and during anti-HIV therapy in persons with acute, chronic, and late-stage HIV-1 infection. Among seven individuals treated with anti-retroviral therapy (ART) and four individuals deferring therapy we found evidence of significant paracortical T cell zone damage associated with deposition of collagen, the extent of which was inversely correlated with both the size of the LT CD4⁺ T cell population and the change in peripheral CD4⁺ T cell count with anti-HIV therapy. The HIV-1–associated inflammatory changes and scarring in LT both limit the ability of the tissue to support and reestablish normal-sized populations of CD4⁺ T cells and suggest a novel mechanism of T cell depletion that may explain the failure of ART to significantly increase CD4⁺ T cell populations in some HIV-1–infected persons.

E rythropoietin-producing hepatocyte (Eph) kinases represent the largest receptor tyrosine kinase family. Some of them are expressed in the T cell compartment, but their function in T cells is unknown. In peripheral blood, EphB6 was predominantly expressed on T cells, and was upregulated after culture. EphB6 crosslinking by anti-EphB6 mAb or ephrinB2 in the presence of suboptimal T cell receptor (TCR) stimulation led to drastic T cell proliferation, suggesting that EphB6 can co-stimulate T cells. The proliferation was accompanied by enhanced production of several lymphokines, such as IFN-γ, IL-6, IL-10, TGF-β, TNF-α, and GM-CSF, but not IL-2 and IL-4. Sorted EphB6⁺ T cells had significantly stronger response to anti-CD3 and anti-CD28 stimulation than EphB6^– T cells had. Taken together, these data suggest an important role of EphB6 in normal T cell activation. Within two minutes of anti-CD3 and anti-CD28 stimulation, EphB6 aggregated and colocalized with TCR, and this provides a morphological basis for EphB6 to enhance TCR signaling. The capping was followed by p38 MAPK activation, showing that EphB6 is capable of signaling, in spite of its lack of intrinsic kinase activity. This study demonstrates that interaction between EphB6 and its ligands facilitates T cell responses to antigen.

T o gain a better understanding of bacterial responses to complex and hostile environments generated within the neutrophil phagosome, we estimated mRNA abundance, using genomic arrays, in Escherichia coli cells ingested by normal and phagocyte oxidase-deficient human neutrophils. Genes regulated by the oxidant sensing transcription factor OxyR were among those strongly induced upon phagocytosis by normal, but not oxidase-deficient, neutrophils. Several genes related to nitrogen metabolism, especially those regulated by the NtrC and NAC proteins and transcribed via the ς⁵⁴ alternative sigma factor, were suppressed by both normal and oxidase-deficient neutrophils. A ΔoxyRS mutant strain of E. coli was significantly more susceptible than the parent strain to neutrophil-mediated killing, which suggests that OxyR-regulated gene products contribute a measure of resistance to neutrophil antimicrobial systems. The hypersusceptibility of the ΔoxyRS mutant was attenuated when oxidase-deficient neutrophils were employed, suggesting that much of the protection afforded by the OxyR regulon is against oxidative antimicrobial factors. Expression profiling of phagocytosed bacteria appears to provide useful information about conditions in the phagocytic vacuole and about bacterial defenses mounted in response to this hostile environment.

W e investigated the effect of Notch signaling, a known regulator of cell fate in numerous developmental systems, on human hematopoietic precursors. We show that activation of endogenous Notch signaling in human CD34⁺CD38^– cord blood precursors with immobilized Delta-1 in serum-free cultures containing fibronectin and hematopoietic growth factors inhibited myeloid differentiation and induced a 100-fold increase in the number of CD34⁺ cells compared with control cultures. Immobilized Delta-1 also induced a multifold expansion of cells with the phenotype of common lymphoid precursors (CD34⁺CD7⁺CD45RA⁺) and promoted the development of cytoplasmic CD3⁺ T/NK cell precursors. IL-7 enhanced the promotion of T/NK cell differentiation by immobilized Delta-1, but granulocytic differentiation occurred when G-CSF was added. Transplantation into immunodeficient mice showed a substantial increase in myeloid and B cell engraftment in the marrow and also revealed thymic repopulation by CD3⁺ T cells due to cells being cultured for a longer period with immobilized Delta-1. These data suggest that Delta-1 can enhance myeloid and lymphoid marrow-repopulating ability and promote the generation of thymus-repopulating T cell precursors.

T he adaptive immune response is triggered by recognition of T and B cell epitopes and is influenced by “danger” motifs that act via innate immune receptors. This study shows that motifs associated with noncoding RNA are essential features in the immune response reminiscent of viral infection, mediating rapid induction of proinflammatory chemokine expression, recruitment and activation of antigen-presenting cells, modulation of regulatory cytokines, subsequent differentiation of Th1 cells, isotype switching, and stimulation of cross-priming. The heterogeneity of RNA-associated motifs results in differential binding to cellular receptors, and specifically impacts the immune profile. Naturally occurring double-stranded RNA (dsRNA) triggered activation of dendritic cells and enhancement of specific immunity, similar to selected synthetic dsRNA motifs. Based on the ability of specific RNA motifs to block tolerance induction and effectively organize the immune defense during viral infection, we conclude that such RNA species are potent danger motifs. We also demonstrate the feasibility of using selected RNA motifs as adjuvants in the context of novel aerosol carriers for optimizing the immune response to subunit vaccines. In conclusion, RNA-associated motifs produced during viral infection bridge the early response with the late adaptive phase, regulating the activation and differentiation of antigen-specific B and T cells, in addition to a short-term impact on innate immunity.

M any painful inflammatory and ischemic conditions such as rheumatoid arthritis, cardiac ischemia, and exhausted skeletal muscles are accompanied by local tissue acidosis. In such acidotic states, extracellular protons provoke the pain by opening cation channels in nociceptors. It is generally believed that a vanilloid receptor subtype-1 (VR1) and an acid-sensing ion channel (ASIC) mediate the greater part of acid-induced nociception in mammals. Here we provide evidence for the involvement of both channels in acid-evoked pain in humans and show their relative contributions to the nociception. In our psychophysical experiments, direct infusion of acidic solutions (pH ≥ 6.0) into human skin caused localized pain, which was blocked by amiloride, an inhibitor of ASICs, but not by capsazepine, an inhibitor of VR1. Under more severe acidification (pH 5.0) amiloride was less effective in reducing acid-evoked pain. In addition, capsazepine had a partial blocking effect under these conditions. Amiloride itself neither blocked capsaicin-evoked localized pain in human skin nor inhibited proton-induced currents in VR1-expressing Xenopus oocytes. Our results suggest that ASICs are leading acid sensors in human nociceptors and that VR1 participates in the nociception mainly under extremely acidic conditions.

C holesterol is converted into dozens of primary and secondary bile acids through pathways subject to negative feedback regulation mediated by the nuclear receptor farnesoid X receptor (FXR) and other effectors. Disruption of the sterol 12α-hydroxylase gene (Cyp8b1) in mice prevents the synthesis of cholate, a primary bile acid, and its metabolites. Feedback regulation of the rate-limiting biosynthetic enzyme cholesterol 7α-hydroxylase (CYP7A1) is lost in Cyp8b1^–/– mice, causing expansion of the bile acid pool and alterations in cholesterol metabolism. Expression of other FXR target genes is unaltered in these mice. Cholate restores CYP7A1 regulation in vivo and in vitro. The results implicate cholate as an important negative regulator of bile acid synthesis and provide preliminary evidence for ligand-specific gene activation by a nuclear receptor.

T he function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na⁺ channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (ΔK1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and ΔK1500 mutant channel. The mutation shifted V_1/2h_∞ to more negative membrane potentials and increased k_h consistent with a reduction of inactivation valence of 1. The shift in h_∞ was the result of an increase in closed-state inactivation rate (11-fold at –100 mV). V_1/2m was shifted to more positive potentials, and k_m was doubled in the ΔK1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (change in charge, –1 and –2, respectively). For both mutations, V_1/2h was shifted back toward control; however, V_1/2m shifted progressively to more positive potentials. The late component of Na⁺ current was increased in the ΔK1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation.