Issue published July 1, 2000

C erebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase–deficient (eNOS-deficient; eNOS^–/–) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion. We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion. Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 μg/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity. This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 ± 3 hours to 24 ± 4 hours. Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression. Inhibition of vascular Rho guanosine-5′-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion. No neuroprotection was observed with these agents in eNOS^–/– mice. These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

C linical and histologic similarities between various eczematous disorders point to a common efferent pathway. We demonstrate here that activated T cells infiltrating the skin in atopic dermatitis (AD) and allergic contact dermatitis (ACD) induce keratinocyte (KC) apoptosis. KCs normally express low levels of Fas receptor (FasR) that can be substantially enhanced by the presence of IFN-γ. KCs are rendered susceptible to apoptosis by IFN-γ when FasR numbers reach a threshold of approximately 40,000 per KC. Subsequently, KCs undergo apoptosis induced by anti-FasR mAb’s, soluble Fas ligand, supernatants from activated T cells, or direct contact between T cells and KCs. Apoptotic KCs show typical DNA fragmentation and membrane phosphatidylserine expression. KC apoptosis was demonstrated in situ in lesional skin affected by AD, ACD, and patch tests. Using numerous cytokines and anti-cytokine neutralizing mAb’s, we found no evidence that cytokines other than IFN-γ participate in this process. In addition, apoptosis-inducing pathways other than FasR triggering were ruled out by blocking T cell–induced KC apoptosis by caspase inhibitors and soluble Fas-Fc protein. Responses of normal human skin and cultured skin equivalents to activated T cells demonstrated that KC apoptosis caused by skin-infiltrating T cells is a key event in the pathogenesis of eczematous dermatitis.

S imian immunodeficiency virus (SIV) infection of rhesus monkeys provides an excellent model of the central nervous system (CNS) consequences of HIV infection. To discern the relationship between viral load and abnormalities induced in the CNS by the virus, we infected animals with SIV and later instituted antiviral treatment to lower peripheral viral load. Measurement of sensory-evoked potentials, assessing CNS neuronal circuitry, revealed delayed latencies after infection that could be reversed by lowering viral load. Cessation of treatment led to the reappearance of these abnormalities. In contrast, the decline in general motor activity induced by SIV infection was unaffected by antiviral treatment. An acute increase in the level of the chemokine monocyte chemoattractant protein-1 (MCP-1) was found in the cerebrospinal fluid (CSF) relative to plasma in the infected animals at the peak of acute viremia, likely contributing to an early influx of immune cells into the CNS. Examination of the brains of the infected animals after return of the electrophysiological abnormalities revealed diverse viral and inflammatory findings. Although some of the physiological abnormalities resulting from SIV infection can be at least temporarily reversed by lowering viral load, the viral-host interactions initiated by infection may result in long-lasting changes in CNS-mediated functions.

H LA-B27 is highly associated with ankylosing spondylitis (AS), but the mechanism is unknown. Among the HLA-B27 alleles, B*2709, which differs by one amino acid from the susceptible B*2705, is not associated with the disease. Here, we analyze the reactivity, in patients with AS and in healthy controls carrying the B*2709 or B*2705 alleles, to an EBV epitope derived from LMP2 (236-244) and to a sequence-related self-peptide from vasoactive intestinal peptide receptor 1 (VIP1R 400-408). We found that both B*2705⁺ and B*2709⁺ subjects possess LMP2 236-244–specific, HLA-B27–restricted T cells, whereas only the B*2705⁺ individuals respond significantly to VIP1R 400-408. These results prompted us to compare, by IFN-γ ELISPOT analysis, the T-cell response to VIP1R 400-408 in patients with AS versus B*2705 healthy controls. The data show that VIP1R 400-408–specific reactivity is a major feature of the patients with AS. These findings show, for the first time to our knowledge, a widespread reactivity in patients with AS against a self-epitope that exhibits some features of a putative “arthritogenic” peptide.

M atrix metalloproteinase-9 (MMP-9) is prominently overexpressed after myocardial infarction (MI). We tested the hypothesis that mice with targeted deletion of MMP9 have less left ventricular (LV) dilation after experimental MI than do sibling wild-type (WT) mice. Animals that survived ligation of the left coronary artery underwent echocardiographic studies after MI; all analyses were performed without knowledge of mouse genotype. By day 8, MMP9 knockout (KO) mice had significantly smaller increases in end-diastolic and end-systolic ventricular dimensions at both midpapillary and apical levels, compared with infarcted WT mice; these differences persisted at 15 days after MI. MMP-9 KO mice had less collagen accumulation in the infarcted area than did WT mice, and they showed enhanced expression of MMP-2, MMP-13, and TIMP-1 and a reduced number of macrophages. We conclude that targeted deletion of the MMP9 gene attenuates LV dilation after experimental MI in mice. The decrease in collagen accumulation and the enhanced expression of other MMPs suggest that MMP-9 plays a prominent role in extracellular matrix remodeling after MI.

W e used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (–/–) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4^–/– mice have impaired Th1 responses, whereas STAT6^–/– mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4^–/–, and STAT6^–/– BALB/c recipients. STAT4^–/– and STAT6^–/– mice rejected their grafts with the same tempo as untreated WT recipients. CD28-B7 blockade by a single injection of CTLA4Ig induced long-term engraftment and donor-specific tolerance in all three groups of recipients. CD154 blockade by a single injection of MR1 was effective in prolonging allograft survival and inducing tolerance in STAT4^–/– mice but was only marginally effective in STAT6^–/– recipients and WT controls. In addition, a similar protocol of MR1 was ineffective in prolonging graft survival in CD28^–/– BALB/c recipients, suggesting that the lack of efficacy seen in WT and STAT6^–/– mice is not due to the presence of a functional CD28-B7 pathway. Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund’s adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation pathways in vivo and may have potential implications for the development of therapeutic strategies such as T-cell costimulatory blockade in humans.

V itamin A and retinoids affect pituitary-thyroid function through suppression of serum thyroid-stimulating hormone (TSH) levels and TSH-β subunit gene expression. We have previously shown that retinoid X receptor–selective (RXR-selective) ligands can suppress serum TSH levels in vivo and TSH-β promoter activity in vitro. The RXR-γ isotype has limited tissue distribution that includes the thyrotrope cells of the anterior pituitary gland. In this study, we have performed a detailed analysis of the pituitary-thyroid function of mice lacking the gene for the RXR-γ isotype. These mice had significantly higher serum T4 levels and TSH levels than did wild-type (WT) controls. Treatment of RXR-γ–deficient and WT mice with T3 suppressed serum TSH and T4 levels in both groups, but RXR-γ–deficient mice were relatively resistant to exogenous T3. RXR-γ–deficient mice had significantly higher metabolic rates than did WT controls, suggesting that these animals have a pattern of central resistance to thyroid hormone. RXR-γ, which is also expressed in skeletal muscle and the hypothalamus, may have a direct effect on muscle metabolism, regulation of food intake, or thyrotropin-releasing hormone levels in the hypothalamus. In conclusion, the RXR-γ isotype appears to contribute to the regulation of serum TSH and T4 levels and to affect peripheral metabolism through regulation of the hypothalamic-pituitary-thyroid axis or through direct effects on skeletal muscle.

R odents given a supramaximally stimulating dose of cholecystokinin or its analogue cerulein develop acute pancreatitis with acinar cell injury, pancreatic inflammation, and intrapancreatic digestive enzyme (i.e., trypsinogen) activation. Prior thermal stress is associated with heat shock protein 70 (HSP70) expression and protection against cerulein-induced pancreatitis. However, thermal stress can also induce expression of other HSPs. The current studies were performed using an in vitro system to determine whether HSP70 can actually mediate protection against pancreatitis and, if so, to define the mechanism underlying that protection. We show that in vitro exposure of freshly prepared rat pancreas fragments to a supramaximally stimulating dose of cerulein results in changes similar to those noted in cerulein-induced pancreatitis, i.e., intra-acinar cell trypsinogen activation and acinar cell injury. Short-term culture of the fragments results in HSP70 expression and loss of the pancreatitis-like changes noted after addition of cerulein. The culture-induced enhanced HSP70 expression can be prevented by addition of either the flavonoid antioxidant quercetin or an antisense oligonucleotide to HSP70. Under these latter conditions, addition of a supramaximally stimulating concentration of cerulein results in trypsinogen activation and acinar cell injury. These findings indicate that the protection against cerulein-induced pancreatitis that follows culture-induced (and possibly thermal) stress is mediated by HSP70. They suggest that the HSP acts by preventing trypsinogen activation within acinar cells.

L ong-term administration of CTLA4Ig prevents the onset of disease in systemic lupus erythematosus–prone (SLE-prone) NZB/NZW F1 mice. To determine the mechanism of this effect, we engineered an adenovirus that expresses murine CTLA4Ig. Administration of a single high dose of this virus results in long-term expression of CTLA4Ig in the serum and absence of an immune response to the adenoviral vector. We administered Ad-CTLA4Ig to 19- to 22-week-old NZB/NZW F1 mice and evaluated the effect on anti-DNA antibody–producing B cells. We show that CTLA4Ig has a beneficial effect on murine SLE for as long as it is present in the serum. This effect is associated with decreased expansion of both the IgM and IgG autoreactive B-cell population, inhibition of immunoglobulin class switching, decreased frequency and altered pattern of somatic mutation, and a marked decrease in the numbers of activated CD4-positive T cells. In contrast, intrinsic B-cell hyperreactivity and the survival of plasma cells in the bone marrow, both of which are less dependent on T-cell help, appear to be unaffected by CTLA4Ig. High-dose CTLA4Ig did not induce permanent tolerance in this autoimmune disease model. Furthermore, although the mice survived in a conventional housing facility, treatment with Ad-CTLA4Ig was immunosuppressive.

T he renin-angiotensin system (RAS) plays a critical role in cardiovascular and fluid homeostasis. The major biologically active peptide of the RAS is angiotensin II, which acts through G protein–coupled receptors of two pharmacological classes, AT₁ and AT₂. AT₁ receptors, expressed in brain and peripheral tissues, mediate most classically recognized actions of the RAS, including blood pressure homeostasis and regulation of drinking and water balance. In rodents, two highly homologous AT₁ receptor isoforms, termed AT_1A and AT_1B receptors, are expressed at different levels in major forebrain cardiovascular and fluid regulatory centers, with AT_1A expression generally exceeding AT_1B expression, but the relative contributions of these receptor subtypes to central angiotensin II responses are not known. We used gene targeting in combination with a unique system for maintaining catheters in the cerebral ventricles of conscious mice to test whether there are differential roles for AT_1A and AT_1B receptors in responses elicited by angiotensin II in the brain. Here we show that the blood pressure increase elicited by centrally administered angiotensin II can be selectively ascribed to the AT_1A receptor. However, the drinking response requires the presence of AT_1B receptors. To our knowledge, this is the first demonstration of a primary and nonredundant physiological function for AT_1B receptors.

V asopressin and its analogue 1-deamino-8-D-arginine vasopressin (DDAVP) are known to raise plasma von Willebrand factor (vWF) levels. DDAVP is used as a hemostatic agent for the treatment of von Willebrand’s disease. However, its cellular mechanisms of action have not been elucidated. DDAVP, a specific agonist for the vasopressin V2 receptor (V2R), exerts its antidiuretic effect via a rise in cAMP in kidney collecting ducts. We tested the hypothesis that DDAVP induces vWF secretion by binding to V2R and activating cAMP-mediated signaling in endothelial cells. vWF secretion from human umbilical vein endothelial cells (HUVECs) can be mediated by cAMP, but DDAVP is ineffective, presumably due to the absence of V2R. We report that DDAVP stimulates vWF secretion in a cAMP-dependent manner in HUVECs after transfection of the V2R. In addition, vasopressin and DDAVP induce vWF secretion in human lung microvascular endothelial cells (HMVEC-L). These cells (but not HUVECs) express endogenous V2R, as shown by RT-PCR. Vasopressin-induced vWF secretion is mimicked by DDAVP and inhibited by the selective V2R antagonist SR121463B. It is mediated by cAMP, since it is inhibited by the protein kinase A inhibitor Rp-8CPT-cAMPS. These results indicate that vasopressin induces cAMP-mediated vWF secretion by a direct effect on endothelial cells. They also demonstrate functional expression of V2R in endothelial cells, and provide a cellular mechanism for the hemostatic effects of DDAVP.

T he continuous, in vivo infusion of low-dose IL-2 selectively expands the absolute number of human natural killer (NK) cells after 4–6 weeks of therapy. The mechanism responsible for this expansion is unknown and was examined in this study. NK cells cultured at low concentrations of IL-2, comparable to those found during in vivo therapy, proliferate for 6 days and then exit the cell cycle. However, NK cells in vivo did not traverse the S/G₂/M phase of the cell cycle during low-dose IL-2 therapy. Low concentrations of IL-2 delay programmed cell death of NK cells but have the same effect on resting T cells that do not expand in vivo. When CD34⁺ bone marrow hematopoietic progenitor cells are cultured for 21 days with low concentrations of IL-2, they differentiate into CD56⁺CD3⁻ NK cells, not T cells. Thus, the selective expansion of human NK cells during continuous in vivo infusion of low-dose IL-2 likely results from enhanced NK-cell differentiation from bone marrow progenitors, combined with an IL-2–dependent delay in NK-cell death, rather than proliferation of mature NK cells in the periphery.

T he aim of the present study was to develop a chronic in vivo model of pulmonary β₂-adrenoceptor desensitization and to elucidate the nature and molecular basis of this state. Subcutaneous infusion of rats with albuterol for 7 days compromised the ability of albuterol, given acutely, to protect against acetylcholine-induced bronchoconstriction. The bronchoprotective effect of prostaglandin E₂, but not forskolin, was also impaired, indicating that the desensitization was heterologous and that the primary defect in signaling was upstream of adenylyl cyclase. β₂-Adrenoceptor density was reduced in lung membranes harvested from albuterol-treated animals, and this was associated with impaired albuterol-induced cyclic adenosine monophosphate (cAMP) accumulation and activation of cAMP-dependent protein kinase ex vivo. Gsα expression was reduced in the lung and tracheae of albuterol-treated rats, and cholera toxin–induced cAMP accumulation was blunted. Chronic treatment of rats with albuterol also increased cAMP phosphodiesterase activity and G protein–coupled receptor kinase-2, but the extent to which these events contributed to β₂-adrenoceptor desensitization was unclear given that forskolin was active in both groups of animals and that desensitization was heterologous. Collectively, these results indicate that albuterol effects heterologous desensitization of pulmonary Gs-coupled receptors in this model, with downregulation of Gsα representing a primary molecular etiology.

I n an attempt to isolate disease-associated autoantigens in rheumatoid arthritis (RA), we cloned a new autoantigen named gp130-RAPS, which is a novel soluble form of the IL-6 signal–transducing molecule gp130. gp130-RAPS is a 50-kDa protein translated from alternatively spliced mRNA and has a truncated form of gp130 with a unique sequence, Asn-Ile-Ala-Ser-Phe (NIASF), in its COOH-terminus. We observed serum antibodies to this NIASF sequence frequently in patients with RA, but not in those with other systemic rheumatic diseases or in healthy subjects. In RA, detection of those antibodies was significantly associated with disease activity indices such as serum C–reactive protein (CRP) levels, erythrocyte sedimentation rate, blood platelet counts, and serum IL-6 concentration. In vitro experiments revealed that gp130-RAPS inhibited IL-6 activity, and this inhibition was neutralized by antibodies to the COOH-terminus of gp130-RAPS derived from patients with RA. Thus, autoantibody to gp130-RAPS may play an important role in the progression of RA by promoting IL-6 activity. Inspection of autoantibodies to gp130-RAPS may become a practical clinical test for RA. gp130-RAPS and its autoantibody provide a new clue to the complicated pathogenesis of RA.

T he ultimate goal of transplantation is drug-free allograft acceptance, which is rarely encountered in transplant recipients. Using a novel human-to-mouse “trans vivo” delayed-type hypersensitivity assay, we assessed donor-reactive cell-mediated immune responses in kidney and liver transplant patients, four of whom discontinued all immunosuppression. One of these subjects (J.B.) rejected his graft after 7 years of stable function, while the others (D.S., R.D., M.L.) continue to have excellent graft function 5, 28, and 4 years after the cessation of immunosuppression. PBMCs from J.B. exhibited strong responses to both donor and recall antigens whereas PBMCs from patients D.S., R.D., and M.L. responded strongly to recall, but not donor, antigens. Furthermore, when donor and recall antigens were colocalized, the recall response in these three patients was inhibited. This donor antigen–linked nonresponsiveness was observed in four other patients who are still maintained on immunosuppression. The weakness of donor-reactive DTH responses in these patients is due to donor alloantigen-triggered regulation that relies on either TGF-β or IL-10. In D.S., regulation is triggered by a single donor HLA Class I antigen, either in membrane-bound or soluble form. This demonstrates that allograft acceptance in humans is associated with an immune regulation pattern, which may be useful in the diagnosis and/or monitoring of transplant patients for allograft acceptance.