Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of lipocortin 1

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5–6 cells per 100-μm of vessel length) and emigration (maximal at 4 hr: 8–10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2–26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (≥50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2–26 (13 mg/kg) or recombinant human LC1 (0.7–2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ± 0.75 min and 2.36 ± 0.31 min, respectively (n = 20–25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.

Agonist-selective endocytosis of mu opioid receptor by neurons in vivo.

Opiate alkaloids are potent analgesics that exert multiple pharmacological effects in the nervous system by activating G protein-coupled receptors. Receptor internalization upon stimulation may be important for desensitization and resensitization, which affect cellular responsiveness to ligands. Here, we investigated the agonist-induced internalization of the mu opioid receptor (MOR) in vivo by using the guinea pig ileum as a model system and immunohistochemistry with an affinity-purified antibody to the C terminus of rat MOR. Antibody specificity was confirmed by the positive staining of human embryonic kidney 293 cells transfected with epitope-tagged MOR cDNA, by the lack of staining of cells transfected with the delta or kappa receptor cDNA, and by the abolition of staining when the MOR antibody was preadsorbed with the MOR peptide fragment. Abundant MOR immunoreactivity (MOR-IR) was localized to the cell body, dendrites, and axonal processes of myenteric neurons. Immunostaining was primarily confined to the plasma membrane of cell bodies and processes. Within 15 min of an intraperitoneal injection of the opiate agonist etorphine, intense MOR-IR was present in vesicle-like structures, which were identified as endosomes by confocal microscopy. At 30 min, MOR-IR was throughout the cytoplasm and in perinuclear vesicles. MOR-IR was still internalized at 120 min. Agonist-induced endocytosis was completely inhibited by the opiate antagonist naloxone. Interestingly, morphine, a high-affinity MOR agonist, did not cause detectable internalization, but it partially inhibited the etorphine-induced MOR endocytosis. These results demonstrate the occurrence of agonist-selective MOR endocytosis in neurons naturally expressing this receptor in vivo and suggest the existence of different mechanisms regulating cellular responsiveness to ligands.

Hippocampal acetylcholine release during memory testing in rats: augmentation by glucose.

Several lines of evidence indicate that a modest increase in circulating glucose levels enhances memory. One mechanism underlying glucose effects on memory may be an increase in acetylcholine (ACh) release. The present experiment determined whether enhancement of spontaneous alternation performance by systemic glucose treatment is related to an increase in hippocampal ACh output. Samples of extracellular ACh were assessed at 12-min intervals using in vivo microdialysis with HPLC-EC. Twenty-four minutes after an intraperitoneal injection of saline or glucose (100, 250, or 1000 mg/kg), rats were tested in a four-arm cross maze for spontaneous alternation behavior combined with microdialysis collection. Glucose at 250 mg/kg, but not 100 or 1000 mg/kg, produced an increase in spontaneous alternation scores (69.5%) and ACh output (121.5% versus baseline) compared to alternation scores (44.7%) and ACh output (58.9% versus baseline) of saline controls. The glucose-induced increase in alternation scores and ACh output was not secondary to changes in locomotor activity. Saline and glucose (100-1000 mg/kg) treatment had no effect on hippocampal ACh output when rats remained in the holding chamber. These findings suggest that glucose may enhance memory by directly or indirectly increasing the release of ACh. The results also indicate that hippocampal ACh release is increased in rats performing a spatial task. Moreover, because glucose enhanced ACh output only during behavioral testing, circulating glucose may modulate ACh release only under conditions in which cholinergic cells are activated.

Anti-nuclear antibody production and immune-complex glomerulonephritis in BALB/c mice treated with pristane.

The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.

Correlation of peptide specificity and IgG subclass with pathogenic and nonpathogenic autoantibodies in pemphigus vulgaris: a model for autoimmunity.

Pemphigus vulgaris (PV) is a rare, potentially fatal, autoimmune disease that affects the skin and mucous membranes. The PV antigen (PVA) has been characterized as desmoglein 3. PV patients carry HLA-DR4- or HLA-DR6-bearing extended haplotypes. We recently demonstrated that patients with active disease have high titers of PV autoantibodies of the IgG1 and IgG4 subclasses. Patients in remission, healthy unaffected relatives, and some MHC-matched normal individuals have low levels of PV autoantibodies, which are IgG1 only. Furthermore, intraperitoneal injection of IgG from patients with active disease caused clinical disease in mice, but IgG from patients in remission, healthy relatives, or MHC-matched normal individuals did not. We prepared 12 peptides of 30 amino acids each (peptides Bos 1-12) spanning the extracellular domain of PVA. Patients with active disease recognize peptides Bos 1 and Bos 6 with high titers of IgG1 and IgG4 autoantibodies. Patients in remission have IgG1 autoantibodies to peptide Bos 1 only, in statistically significantly lower titers (P < 0.01). They no longer have IgG4 subclass autoantibodies to peptide Bos 6. Healthy relatives and normal unrelated individuals have low levels of only IgG1 autoantibodies that recognize only Bos 1. In vitro studies indicate that Bos 6-specific IgG and, to a lesser extent, Bos 1-specific IgG can cause acantholysis. Our data suggest that Bos 6-specific IgG4 is probably the main acantholytic autoantibody, while Bos 1-specific IgG4 may act as a facilitator or enhancer of the process. In this study we illustrate some of the paradigms that demonstrate the interactions between the MHC, subclass of autoantibodies, and peptide specificities of the autoantibodies in the autoimmune process. Thus, PV provides an important model to study the pathogenesis of autoimmunity.

Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat 5 in mouse liver.

Intraperitoneal injection of epidermal growth factor into mice results in the appearance of multiple tyrosine-phosphorylated proteins in liver nuclei within minutes after administration. We have previously identified three of these proteins as Stat 1 alpha, Stat 1 beta (p91, p84), and Stat 3 (p89). In the present report we demonstrate that Stat 5 (p92), the recently described prolactin inducible transcription factor detected in mammary glands, is the major tyrosine-phosphorylated protein translocated to the nucleus in mouse liver in response to epidermal growth factor. Furthermore, gel-shift analysis and affinity purification revealed that Stat 5, Stat 1 alpha, and Stat 1 beta specifically bind to the prolactin inducible element upstream of the beta-casein promoter.

Pig but not human interferon-γ initiates human cell-mediated rejection of pig tissue in vivo

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ) induced human CD4+ and CD8+ T cells and macrophages to more extensivley infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.

Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

Induction of inhibitory factor κBα mRNA in the central nervous system after peripheral lipopolysaccharide administration: An in situ hybridization histochemistry study in the rat

In this study we investigate the mRNA expression of inhibitory factor κBα (IκBα) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). IκB controls the activity of nuclear factor κB, which regulates the transcription of many immune signal molecules. The detection of IκB induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of IκBα mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of IκBα mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1–2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, IκBα mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood–brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood–brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.

Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2

CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines. Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation. Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice. However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was ≈3.5 times the basal level. The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils. However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages. After intravenous injection with yeast β-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas. These results demonstrate that CCR2 is a major regulator of induced macrophage trafficking in vivo.

Signal changes in the spinal cord of the rat after injection of formalin into the hindpaw: Characterization using functional magnetic resonance imaging

Changes in metabolism and local circulation occur in the spinal cord during peripheral noxious stimulation. Evidence is presented that this stimulation also causes signal intensity alterations in functional magnetic resonance images of the spinal cord during formalin-induced pain. These results indicate the potential of functional magnetic resonance imaging in assessing noninvasively the extent and intensity of spinal cord excitation in this well characterized pain model. Therefore, the aim of this study was to establish functional magnetic resonance imaging as a noninvasive method to characterize temporal changes in the spinal cord after a single injection of 50 μl of formalin subcutaneously into the hindpaw of the anesthetized rat. This challenge produced a biphasic licking activity in the freely moving conscious animal. Images of the spinal cord were acquired within 2 min, enabling monitoring of the site and the temporal evolution of the signal changes during the development of formalin-induced hyperalgesia without the need of any surgical procedure. The time course of changes in the spinal cord functional image in the isoflurane-anesthetized animal was similar to that obtained from behavioral experiments. Also, comparable physiological data, control experiments, and the inhibition of a response through application of the local anesthetic agent lidocaine indicate that the signal changes observed after formalin injection were specifically related to excitability changes in the relevant segments of the lumbar spinal cord. This approach could be useful to characterize different models of pain and hyperalgesia and, more importantly, to evaluate effects of analgesic drugs.

In vivo NMR and MRI using injection delivery of laser-polarized xenon

Because xenon NMR is highly sensitive to the local environment, laser-polarized xenon could be a unique probe of living tissues. Realization of clinical and medical science applications beyond lung airspace imaging requires methods of efficient delivery of laser-polarized xenon to tissues, because of the short spin-lattice relaxation times and relatively low concentrations of xenon attainable in the body. Preliminary results from the application of a polarized xenon injection technique for in vivo 129Xe NMR/MRI are extrapolated along with a simple model of xenon transit to show that the peak local concentration of polarized xenon delivered to tissues by injection may exceed that delivered by respiration by severalfold.

Neurosteroids: Deficient cognitive performance in aged rats depends on low pregnenolone sulfate levels in the hippocampus

Pregnenolone sulfate (PREG S) is synthesized in the nervous system and is a major neurosteroid in the rat brain. Its concentrations were measured in the hippocampus and other brain areas of single adult and aged (22–24 month-old) male Sprague–Dawley rats. Significantly lower levels were found in aged rats, although the values were widely scattered and reached, in about half the animals, the same range as those of young ones. The spatial memory performances of aged rats were investigated in two different spatial memory tasks, the Morris water maze and Y-maze. Performances in both tests were significantly correlated and, accompanied by appropriate controls, likely evaluated genuine memory function. Importantly, individual hippocampal PREG S and distance to reach the platform in the water maze were linked by a significant correlation, i.e., those rats with lower memory deficit had the highest PREG S levels, whereas no relationship was found with the PREG S content in other brain areas (amygdala, prefrontal cortex, parietal cortex, striatum). Moreover, the memory deficit of cognitively impaired aged rats was transiently corrected after either intraperitoneal or bilateral intrahippocampal injection of PREG S. PREG S is both a γ-aminobutyric acid antagonist and a positive allosteric modulator at the N-methyl-d-aspartate receptor, and may reinforce neurotransmitter system(s) that decline with age. Indeed, intracerebroventricular injection of PREG S was shown to stimulate acetylcholine release in the adult rat hippocampus. In conclusion, it is proposed that the hippocampal content of PREG S plays a physiological role in preserving and/or enhancing cognitive abilities in old animals, possibly via an interaction with central cholinergic systems. Thus, neurosteroids should be further studied in the context of prevention and/or treatment of age-related memory disorders.

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 μg of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.