Migration of immature mouse DC across resting endothelium is mediated by ICAM-2 but independent of beta2-integrins and murine DC-SIGN homologues

Immature dendritic cells (DC) reside in tissues where they initiate immune responses by taking up foreign antigens. Since DC have a limited tissue half-life, the DC pool in tissues has to be replenished constantly. This implies that precursor/immature DC must be able to cross non-activated endothelium using as yet unknown mechanisms. Here we show that immature, but not mature bone marrow-derived murine DC migrate across resting endothelial monolayers in vitro. We find that endothelial intercellular adhesion molecule-2 (ICAM-2) is a major player in transendothelial migration (TEM) of immature DC, accounting for at least 41% of TEM. Surprisingly, the ICAM-2-mediated TEM was independent of beta2-integrins, the known ICAM-2 ligands, since neither blocking of beta2-integrins with antibodies nor the use of CD18-deficient DC affected the ICAM-2-specific TEM. In humans, the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) was shown to interact with ICAM-2, suggesting a similar role in mice. However, we find that none of the murine DC-SIGN homologues mDC-SIGN, murine DC-SIGN-related molecule-1 (mSIGN-R1) and mSIGN-R3 is expressed on the surface of bone marrow-derived mouse DC. Taken together, this study shows that ICAM-2 strongly supports transmigration of immature DC across resting endothelium by interacting with ligands that are distinct from beta2-integrins and DC-SIGN homologues.

P-glycoprotein expression in lamina propria lymphocytes of duodenal biopsy samples in dogs with chronic idiopathic enteropathies

P-glycoprotein (p-gp) is a transmembrane protein functioning as a drug-efflux pump in the intestinal epithelium. Human patients with inflammatory bowel disease (IBD) who fail to respond to treatment with steroids express high levels of p-gp in lamina propria lymphocytes. The purpose of this study was to investigate p-gp expression in duodenal biopsy samples of dogs with chronic enteropathies and to evaluate the expression of p-gp after treatment with a known inducer of p-gp (prednisolone). Duodenal biopsy samples from 48 dogs were evaluated immunohistochemically with the mouse monoclonal antibody C219 for expression of p-gp in lamina propria lymphocytes. Biopsy samples were available from 15 dogs after treatment with prednisolone and 16 dogs after dietary therapy alone ("elimination diet"). Treatment with prednisolone resulted in an increase in p-gp expression (P=0.005). In contrast, dietary treatment alone produced no significant change in p-gp expression (P=0.59). A low p-gp score before initiation of steroid treatment was significantly associated with a positive response to treatment (P=0.01). These results indicate that lamina propria lymphocyte expression of p-gp is upregulated after prednisolone treatment in dogs with IBD, and that mucosal expression of p-gp may be of value in predicting the response to therapy.

Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE-65 in a mouse model of retinal pigment epithelium damage

Stem cell regeneration of damaged tissue has recently been reported in many different organs. Since the loss of retinal pigment epithelium (RPE) in the eye is associated with a major cause of visual loss - specifically, age-related macular degeneration - we investigated whether hematopoietic stem cells (HSC) given systemically can home to the damaged subretinal space and express markers of RPE lineage. Green fluorescent protein (GFP) cells of bone marrow origin were used in a sodium iodate (NaIO(3)) model of RPE damage in the mouse. The optimal time for adoptive transfer of bone marrow-derived stem cells relative to the time of injury and the optimal cell type [whole bone marrow, mobilized peripheral blood, HSC, facilitating cells (FC)] were determined by counting the number of GFP(+) cells in whole eye flat mounts. Immunocytochemistry was performed to identify the bone marrow origin of the cells in the RPE using antibodies for CD45, Sca-1, and c-kit, as well as the expression of the RPE-specific marker, RPE-65. The time at which bone marrow-derived cells were adoptively transferred relative to the time of NaIO(3) injection did not significantly influence the number of cells that homed to the subretinal space. At both one and two weeks after intravenous (i.v.) injection, GFP(+) cells of bone marrow origin were observed in the damaged subretinal space, at sites of RPE loss, but not in the normal subretinal space. The combined transplantation of HSC+FC cells appeared to favor the survival of the homed stem cells at two weeks, and RPE-65 was expressed by adoptively transferred HSC by four weeks. We have shown that systemically injected HSC homed to the subretinal space in the presence of RPE damage and that FC promoted survival of these cells. Furthermore, the RPE-specific marker RPE-65 was expressed on adoptively transferred HSC in the denuded areas.

Tie2 receptor expression and phosphorylation in cultured cells and mouse tissues

Accumulating experimental evidence indicates that endothelial cell growth and blood vessel morphogenesis are processes that are governed by the activity of specifically expressed receptor tyrosine kinases (RTKs). We have used two new rat monoclonal antibodies (mAbs) to study the expression and phosphorylation of one such receptor, mouse Tie2 (tyrosine kinase that contains immunoglobulin-like loops and epidermal-growth-factor-similar domains 2]), in transfected cells, endothelioma cell lines and mouse tissues. The Tie2 receptor was found to be constitutively autophosphorylated when over-expressed in COS7 cells. In contrast, the endogenous Tie2 protein was not phosphorylated in endothelioma cell lines. However, in these cell lines, Tie2 could be induced to become tyrosine phosphorylated, and this activation was found to be independent of Tie1. Studying Tie2 receptor activity during angiogenesis in mouse development, the receptor was only weakly phosphorylated in the early postnatal mouse brain whereas a stronger activation could be detected in mouse embryos at day 10.5 post coitum.

TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases

Triggering receptor expressed on myeloid cells-1 (TREM-1) potently amplifies acute inflammatory responses by enhancing degranulation and secretion of proinflammatory mediators. Here we demonstrate that TREM-1 is also crucially involved in chronic inflammatory bowel diseases (IBD). Myeloid cells of the normal intestine generally lack TREM-1 expression. In experimental mouse models of colitis and in patients with IBD, however, TREM-1 expression in the intestine was upregulated and correlated with disease activity. TREM-1 significantly enhanced the secretion of relevant proinflammatory mediators in intestinal macrophages from IBD patients. Blocking TREM-1 by the administration of an antagonistic peptide substantially attenuated clinical course and histopathological alterations in experimental mouse models of colitis. This effect was also seen when the antagonistic peptide was administered only after the first appearance of clinical signs of colitis. Hence, TREM-1-mediated amplification of inflammation contributes not only to the exacerbation of acute inflammatory disorders but also to the perpetuation of chronic inflammatory disorders. Furthermore, interfering with TREM-1 engagement leads to the simultaneous reduction of production and secretion of a variety of pro-inflammatory mediators such as TNF, IL-6, IL-8 (CXCL8), MCP-1 (CCL2), and IL-1beta. Therefore, TREM-1 may also represent an attractive target for the treatment of chronic inflammatory disorders.

Distribution of amyloid precursor protein and amyloid-beta immunoreactivity in DBA/2J glaucomatous mouse retinas

PURPOSE: Evidence suggests that altered metabolism of amyloid precursor protein (APP) may play a role in the pathophysiology of retinal ganglion cell (RGC) death in the etiology of glaucoma. The authors sought to determine the distribution of APP and amyloid-beta (Abeta) in DBA/2J glaucomatous mouse retinas. METHODS: The retinas of 3- and 15-month-old DBA/2J mice and C57/BL-6 mice (control group) were fixed with 4% paraformaldehyde and processed for immunohistochemistry. Antibodies used included a polyclonal antibody to the C terminus of Abeta 40 and a polyclonal antibody to the APP ectodomain. Immunohistochemically stained tissue was graded using light microscopy. Distribution and semiquantitative expression of APP and Abeta in young and old glaucomatous and normal retinas were determined and compared. RESULTS: Strong APP and Abeta immunoreactivity was found in the RGC layer, optic nerve, and pia/dura of old DBA/2J retinas, with considerably higher intensity found in the old compared with the young DBA/2J mice. In contrast to glaucomatous mice, the control group did not show any notable age-related difference. CONCLUSIONS: Disruption of the homeostatic properties of secreted APP with consecutive Abeta cytotoxicity might be a contributing factor of ganglion cell loss in glaucomatous mouse retinas.

Transgenic system for conditional induction and rescue of chronic myocardial hibernation provides insights into genomic programs of hibernation

A key energy-saving adaptation to chronic hypoxia that enables cardiomyocytes to withstand severe ischemic insults is hibernation, i.e., a reversible arrest of contractile function. Whereas hibernating cardiomyocytes represent the critical reserve of dysfunctional cells that can be potentially rescued, a lack of a suitable animal model has hampered insights on this medically important condition. We developed a transgenic mouse system for conditional induction of long-term hibernation and a system to rescue hibernating cardiomyocytes at will. Via myocardium-specific induction (and, in turn, deinduction) of a VEGF-sequestering soluble receptor, we show that VEGF is indispensable for adjusting the coronary vasculature to match increased oxygen consumption and exploit this finding to generate a hypoperfused heart. Importantly, ensuing ischemia is tunable to a level at which large cohorts of cardiomyocytes are driven to enter a hibernation mode, without cardiac cell death. Relieving the VEGF blockade even months later resulted in rapid revascularization and full recovery of contractile function. Furthermore, we show that left ventricular remodeling associated with hibernation is also fully reversible. The unique opportunity to uncouple hibernation from other ischemic heart phenotypes (e.g., infarction) was used to determine the genetic program of hibernation; uncovering hypoxia-inducible factor target genes associated with metabolic adjustments and induced expression of several cardioprotective genes. Autophagy, specifically self-digestion of mitochondria, was identified as a key prosurvival mechanism in hibernating cardiomyocytes. This system may lend itself for examining the potential utility of treatments to rescue dysfunctional cardiomyocytes and reverse maladaptive remodeling.

Recombinant major urinary proteins of the mouse in specific IgE and IgG testing

Background: Recombinant allergens are preferred over natural allergen extracts in measuring antibodies. We tested the use of recombinant variants of the major mouse allergen Mus m 1 in detection of mouse-specific antibodies in sera of laboratory animal workers and children. Methods: Six recombinant major urinary proteins (MUPs) were produced and antibody-binding capacity was compared to natural Mus m 1 and to mouse urine extract. In a specific subset, cross-reactivity of MUP with Mus m 1 and between the different recombinant MUPs was determined. Results: For IgE antibodies, MUP8 showed high cross-reactivity with Mus m 1. MUP8-specific IgE was found in 55% of the mouse urine IgE-positive sera. Specific IgG and IgG4 antibodies against natural Mus m 1 correlated strongly with antibodies against recombinant MUP8 and were cross-reactive. IgG4 levels against MUP8 and mouse urine extract correlated, but detection of mouse urine-specific IgG4 in the absence of MUP-specific IgG4 was not uncommon. Cross-reactivity of IgG antibodies between MUP8 and Mus m 1 as well as between the different MUPs was high and inhibition varied between 54 and 99%. Conclusion: The mouse allergen Mus m 1 can be replaced in antibody testing by recombinant MUP8. Other MUPs, except MUP4, are interchangeable with MUP8. However, mouse urine extract showed better detection of both mouse-specific IgE and IgG4 levels. Other components in the mouse urine, like mouse albumin and other yet unidentified components, also induce IgE and IgG(4) antibodies.

[Vasculitis as a reason of chronic headache]

A 13-year-old girl presented to our emergency with a one week history of fever and skin rash and new onset of chorea for the last three days. There was a long standing history of right predominant headache; followed by personality change, fatigue, arthralgia and weight loss over the last few months. Previous investigations by head CT and ophthalmological examination did not explain the symptoms. Further investigations revealed peri- and pancarditis with aortic insufficiency, a renal involvement with elevated creatinin, protein- and hematuria and a hemolytic anemia. Diagnosis of lupus eythematodes was confirmed by high ANA, anti-dsDNS and Anticardiolipin antibodies. Within the first 48 hours after admission there was significant deterioration with reduced vigilance and dysarthria. MRI of the brain and dopplersonography of cerebral vessels showed a complete thrombosis of the right medial cerebral artery with a small net of collaterals, irregularities of the left cerebral artery due to vasculitis and several subacute leftsided ischemias. Immunosuppressive therapy with high-dose corticosteroids and cyclophosphamid together with antithrombotic therapy induced an improvement of neurologic, renal and cardiac function.

Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection

Anti-helminth immunity involves CD4+ T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasite that establishes chronic infection. Polyclonal IgG antibodies, present in naive mice and produced following Hp infection, functioned to limit egg production by adult parasites. Comparatively, affinity-matured parasite-specific IgG and IgA antibodies that developed only after multiple infections were required to prevent adult worm development. These data reveal complementary roles for polyclonal and affinity-matured parasite-specific antibodies in preventing enteric helminth infection by limiting parasite fecundity and providing immune protection against reinfection, respectively. We propose that parasite-induced polyclonal antibodies play a dual role, whereby the parasite is allowed to establish chronicity, while parasite load and spread are limited, likely reflecting the long coevolution of helminth parasites with their hosts.

Quantitative isolation of mouse and human intestinal intraepithelial lymphocytes by elutriation centrifugation

Intestinal intraepithelial lymphocytes (IEL) are specialized subsets of T cells with distinct functional capacities. While some IEL subsets are circulating, others such as CD8alphaalpha TCRalphabeta IEL are believed to represent non-circulating resident T cell subsets [Sim, G.K., Intraepithelial lymphocytes and the immune system. Adv. Immunol., 1995. 58: 297-343.]. Current methods to obtain enriched preparations of intraepithelial lymphocytes are mostly based on Percoll density gradient or magnetic bead-based technologies [Lundqvist, C., et al., Isolation of functionally active intraepithelial lymphocytes and enterocytes from human small and large intestine. J. Immunol. Methods, 1992. 152(2): 253-263.]. However, these techniques are hampered by a generally low yield of isolated cells, and potential artifacts due to the interference of the isolation procedure with subsequent functional assays, in particular, when antibodies against cell surface markers are required. Here we describe a new method for obtaining relatively pure populations of intestinal IEL (55-75%) at a high yield (>85%) by elutriation centrifugation. This technique is equally suited for the isolation and enrichment of intraepithelial lymphocytes of both mouse and human origin. Time requirements for fractionating cell suspensions by elutriation centrifugation are comparable to Percoll-, or MACS-based isolation procedures. Hence, the substantially higher yield and the consistent robust enrichment for intraepithelial lymphocytes, together with the gentle treatment of the cells during elutriation that does not interfere with subsequent functional assays, are important aspects that are in favor of using this elegant technology to obtain unmanipulated, unbiased populations of intestinal intraepithelial lymphocytes, and, if desired, also of pure epithelial cells.

Cardiovascular response to beta-adrenergic blockade or activation in 23 inbred mouse strains

We report the characterisation of 27 cardiovascular-related traits in 23 inbred mouse strains. Mice were phenotyped either in response to chronic administration of a single dose of the beta-adrenergic receptor blocker atenolol or under a low and a high dose of the beta-agonist isoproterenol and compared to baseline condition. The robustness of our data is supported by high trait heritabilities (typically H(2)>0.7) and significant correlations of trait values measured in baseline condition with independent multistrain datasets of the Mouse Phenome Database. We then focused on the drug-, dose-, and strain-specific responses to beta-stimulation and beta-blockade of a selection of traits including heart rate, systolic blood pressure, cardiac weight indices, ECG parameters and body weight. Because of the wealth of data accumulated, we applied integrative analyses such as comprehensive bi-clustering to investigate the structure of the response across the different phenotypes, strains and experimental conditions. Information extracted from these analyses is discussed in terms of novelty and biological implications. For example, we observe that traits related to ventricular weight in most strains respond only to the high dose of isoproterenol, while heart rate and atrial weight are already affected by the low dose. Finally, we observe little concordance between strain similarity based on the phenotypes and genotypic relatedness computed from genomic SNP profiles. This indicates that cardiovascular phenotypes are unlikely to segregate according to global phylogeny, but rather be governed by smaller, local differences in the genetic architecture of the various strains.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

Antibodies trap tissue migrating helminth larvae and prevent tissue damage by driving IL-4Rα-independent alternative differentiation of macrophages

Approximately one-third of the world's population suffers from chronic helminth infections with no effective vaccines currently available. Antibodies and alternatively activated macrophages (AAM) form crucial components of protective immunity against challenge infections with intestinal helminths. However, the mechanisms by which antibodies target these large multi-cellular parasites remain obscure. Alternative activation of macrophages during helminth infection has been linked to signaling through the IL-4 receptor alpha chain (IL-4Rα), but the potential effects of antibodies on macrophage differentiation have not been explored. We demonstrate that helminth-specific antibodies induce the rapid trapping of tissue migrating helminth larvae and prevent tissue necrosis following challenge infection with the natural murine parasite Heligmosomoides polygyrus bakeri (Hp). Mice lacking antibodies (JH (-/-)) or activating Fc receptors (FcRγ(-/-)) harbored highly motile larvae, developed extensive tissue damage and accumulated less Arginase-1 expressing macrophages around the larvae. Moreover, Hp-specific antibodies induced FcRγ- and complement-dependent adherence of macrophages to larvae in vitro, resulting in complete larval immobilization. Antibodies together with helminth larvae reprogrammed macrophages to express wound-healing associated genes, including Arginase-1, and the Arginase-1 product L-ornithine directly impaired larval motility. Antibody-induced expression of Arginase-1 in vitro and in vivo occurred independently of IL-4Rα signaling. In summary, we present a novel IL-4Rα-independent mechanism of alternative macrophage activation that is antibody-dependent and which both mediates anti-helminth immunity and prevents tissue disruption caused by migrating larvae.

Osteopontin and cadherin 11 are novel mediators and drug targets for chronic lung diseases

Chronic lung diseases (CLDs) are a considerable source of morbidity and mortality and are thought to arise from dysregulation of normal wound healing processes. An aggressive, feature of many CLDs is pulmonary fibrosis (PF) and is characterized by excess deposition of extracellular matrix (ECM) proteins from myofibroblasts in airways. However, factors regulating myofibroblast biology are incompletely understood. Proteins in the cadherin family contribute epithelial to mesenchymal transition (EMT), a suggested source of myofibroblasts. Cadherin 11 (CDH11) contributes to developmental and pathologic processes that parallel those seen in PF and EMT. Utilizing Cdh11 knockout (Cdh11 -/-) mice, the goal of this study was to characterize the contribution of CDH11 in the bleomycin model of PF and assess the feasibility of treating established PF. We demonstrate CDH11 in macrophages and airway epithelial cells undergoing EMT in lungs of mice given bleomycin and patients with PF. Endpoints consistent with PF including ECM production and myofibroblast formation are reduced in CDH11-targeted mice given bleomycin. Findings suggesting mechanisms of CDH11-dependent fibrosis include the regulation of the profibrotic mediator TGF-â in alveolar macrophages and CDH11-mediated EMT. The results of this study propose CDH11 as a novel drug target for PF. In addition, another CLD, chronic obstructive pulmonary disease (COPD), is characterized by airway inflammation and destruction. Adenosine, a nucleoside signaling molecule generated in response to cell stress is upregulated in patients with COPD and is suggested to contribute to its pathogenesis. An established model of adenosine-mediated lung injury exhibiting features of COPD is the Ada -/- mouse. Previous studies in our lab suggest features of the Ada -/- phenotype may be secondary to adenosine-dependent expression of osteopontin (OPN). OPN is a protein implicated in a variety of human pathology, but its role in COPD has not been examined. To address this, Ada/Opn -/- mice were generated and endpoints consistent with COPD were examined in parallel with Ada -/- mice. Results demonstrate OPN-mediated pulmonary neutrophilia and airway destruction in Ada -/- mice. Furthermore, patients with COPD exhibit increased OPN in airways which correlate with clinical airway obstruction. These results suggest OPN represents a novel biomarker or therapeutic target for the management of patients with COPD. The importance of findings in this thesis is highlighted by the fact that no pharmacologic interventions have been shown to interfere with disease progression or improve survival rates in patients with COPD or PF.