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Biomedical subjects

B Engelhardt

Publications and source records attributed to B Engelhardt.

At least 19 recordsLinked to original sources
PubMed1997

E- and P-selectin are not involved in the recruitment of inflammatory cells across the blood-brain barrier in experimental autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE) inflammatory cells cross the endothelial blood-brain barrier (BBB) and gain access to the central nervous system (CNS). Here we show that E- and P-selectin are not involved in the recruitment of inflammatory cells across the BBB. Neither expression of E- nor P-selectin is induced in BBB-forming endothelium at any time after initiation of EAE. Some of the inflammatory cells present in the CNS during EAE express ligands for E- or P-selectin. However, anti-E- and P-selectin antibodies influence neither immigration of inflammatory cells across the BBB nor the development of EAE. In general, suppression of E- and P-selectin expression on BBB endothelium is dependent on factors derived from the CNS microenvironment, eg, astrocytes. Our results suggest that during EAE suppression of E- and P-selectin expression on the BBB provides a CNS-specific mechanism to reduce leukocyte recruitment into the CNS.

Animals↗
PubMed1997

Induction of acute inflammatory lung injury by staphylococcal enterotoxin B.

Superantigens stimulate T lymphocytes at high frequency by interacting with appropriate Vbeta segments of the TCR. Challenge of mice with bacterial superantigens such as staphylococcal enterotoxin B (SEB) induces the systemic release of cytokines resulting in septic shock and death of sensitized animals. Analysis of putative pathogenic mechanisms of T cell-dependent septic shock revealed that administration of SEB results in acute inflammatory lung injury characterized by a profound increase in vascular permeability. Injury was associated with marked leukocyte infiltration of the lung and induction of cell adhesion molecules including VCAM-1, ICAM-1, and P-selectin, but not E-selectin. Lung infiltrating leukocytes consisted of granulocytes, mononuclear phagocytes and NK cells with granulocytes representing the major fraction. Consistent with a role of neutrophils as cellular mediators of inflammatory organ injury, we demonstrate activation of circulating granulocytes in mice treated with SEB. When compared with granulocytes of control mice, peripheral blood granulocytes of SEB-treated mice were found to express increased levels of cell surface Mac-1, to down-regulate expression of L-selectin, and to respond with an increased production of toxic oxygen metabolites upon exposure to FMLP. Interestingly, TNF-alpha further enhanced FMLP-induced oxidant production by granulocytes from SEB-treated but not control mice, suggesting that the systemic response to SEB increases granulocyte sensitivity to TNF-alpha-mediated signals. Together, these results suggest that acute inflammatory lung injury may contribute to the pathogenesis of T cell-dependent lethal shock in mice challenged with bacterial superantigens and indicate common pathogenic mechanisms of lung injury induced by a large number of distinct inflammatory stimuli.

Acute Disease↗
PubMed1997

Immunosurveillance modelled in vitro: naive and memory T cells spontaneously migrate across unstimulated microvascular endothelium.

As a model for T cell immigration into non-lymphoid tissue we set up an in vitro assay that would allow us to investigate the phenotype of T lymphocytes from peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) or peripheral blood (PBL) of mice, which were able to spontaneously migrate across unstimulated microvascular endothelium. The transendothelial migrating T cell population was enriched for T lymphocytes expressing a "recently activated/memory' phenotype: LFA-1/CD44/ICAM-1high, but also contained CD45RBhigh and LFA-1low T cells, which in the case of MLN T cells were phenotyped as CD4+ and thus characterized as naive T cells. Transmigrated T cells could be further distinguished from their original populations and from each other by their distinct but heterogeneous expression patterns for L-selectin, alpha 4 beta 7-integrin and PECAM-1. This observation suggests the presence of phenotypically different migratory T cells among MLN, PLN and PBL. Additional studies provided evidence that the capacity to migrate across unstimulated microvascular endothelium was a characteristic of a T cell population that could phenotypically be differentiated from activated T cells. The endothelial cells were found to play an active role in selecting the traversing T cell population, as they controlled the number and phenotype of spontaneously transmigrating T cells. Our studies suggest that the capacity to transmigrate across unstimulated microvascular endothelium and hence to immigrate into non-lymphoid tissue is owned by a phenotypically heterogeneous T cell population, which is enriched for memory T cells but not devoid of naive T cells.

Animals↗
PubMed1996

Lymphocyte targeting of the central nervous system: a review of afferent and efferent CNS-immune pathways.

The central nervous system (CNS) in considered to be an immunological privileged site. However, inflammatory reactions in response to virus infections, in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE) suggest that there are definite connections between the CNS and the immune system. In this review, we examine evidence for afferent and efferent pathways of communication between the CNS and the immune system, the pivotal role of regional lymph nodes in T-cell mediated autoimmune disease of the CNS, and the factors involved in lymphocyte targeting of the CNS. Afferent pathways of lymphatic drainage of the brain are well established in a variety of species, especially rodents. Fluid and antigens appear to drain along perivascular spaces populated by immunocompetent perivascular cells. Drainage pathways connect directly via the cribriform plate to nasal lymphatics and cervical lymph nodes. Soluble antigens draining from the brain induce antibody production in the cervical lymph nodes. Using a model of cryolesion-enhanced EAE, we review the role of lymphatic drainage and cervical lymph nodes in the enhancement of cerebral EAE. If a brain wound in the form of a cryolesion is produced 8 days post inoculation (dpi) of antigen in the induction of acute EAE, there is a 6-fold increase in severity of cerebral EAE by 15 dpi. Removal of the cervical lymph nodes significantly reduces such enhancement of EAE. These findings suggest that drainage of antigens from the brain to the cervical lymph nodes, in the presence of activated lymphocytes in the meninges or CNS, results in an enhanced second wave of lymphocytes targeting the brain. In examining the efferent immune pathway by which lymphocytes home to the CNS, several studies have characterized the phenotype of infiltrating T lymphocytes by the use of immunocytochemistry or FACS analysis. T-cells infiltrating the CNS are recently activated/memory lymphocytes typified by their high expression of CD44, LFA-1 and ICAM-1 and low expression of CD45RB in the mouse. Following the induction of EAE in susceptible mice, ICAM-1 and VCAM-1 are dramatically upregulated on CNS vessels; lymphocytes bind to such vessels via the interaction of their known ligands, LFA-1/Mac-1 and alpha 4-integrins, at least in vitro. It appears that alpha 4-integrin plays a key role in lymphocyte recruitment across the blood-brain barrier and may be a major factor in lymphocyte targeting of the CNS. Definition of factors involved in the afferent and efferent connections between the CNS and the immune system may clarify mechanisms involved in immune privilege of the CNS and may open significant therapeutic opportunities for multiple sclerosis.

Afferent Pathways↗
PubMed1996

ICAM-1, VCAM-1, and MAdCAM-1 are expressed on choroid plexus epithelium but not endothelium and mediate binding of lymphocytes in vitro.

The expression of cell adhesion molecules (CAMs) in the choroid plexus was studied in normal brain and during experimental autoimmune encephalomyelitis (EAE) in the SJL/J mouse during inflammation induced by intracerebral injection of killed Corynebacterium parvum in the C3H/He mouse. Both ICAM-1 and VCAM-1, but not MAdCAM-1, were constitutively expressed on choroid plexus epithelium but not on the fenestrated capillary endothelial cells within the choroid plexus. During EAE, we observed an up-regulation of ICAM-1 and VCAM-1 and de novo expression of MAdCAM-1 on choroid plexus epithelial cells. In contrast, endothelial cells in the choroid plexus were not induced to express any of the investigated CAMs. In in situ hybridization analysis we demonstrated that ICAM-1, VCAM-1, and MAdCAM-1 were locally synthesized and that the amount of their mRNAs increased in the inflamed choroid plexus. In vitro, primary choroid plexus epithelial cells could be induced to express ICAM-1, VCAM-1, and MAdCAM-1 on their surface after treatment with proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1, interferon-gamma, and lipopolysaccharide. To investigate the functional status of the expressed CAMs we performed Stamper-Woodruff binding assays on frozen sections of inflamed and naive brains. ICAM-1, VCAM-1, and MAdCAM-1 expressed in choroid plexus epithelial cells mediated binding of lymphocytes via their known ligands LFA-1 and alpha4-integrin, respectively. The expression of ICAM-1, VCAM-1, and MAdCAM-1 on choroid plexus epithelial cells together with the lack of their expression on the fenestrated choroid plexus endothelium raises the possibility that the epithelial blood-cerebrospinal-fluid barrier plays an important role in the immunosurveillance of the central nervous system.

Animals↗
PubMed1995

Lymphocytes infiltrating the CNS during inflammation display a distinctive phenotype and bind to VCAM-1 but not to MAdCAM-1.

The nature of inflammatory lymphocytes recruited to the CNS has been studied in a model of chronic inflammation. Injection of killed Corynebacterium parvum into the cortex of the mouse brain produces a circumscribed inflammatory cellular infiltrate around the injection site, and recruited mononuclear inflammatory cells (IC) can be isolated for flow cytometric analysis. The majority of IC were T cells. In comparison with the predominant naive population of mesenteric lymph node T cells, IC T cells express much higher levels of CD44, LFA-1 and ICAM-1, and lower levels of CD45RB, features commonly associated with memory (previously activated) cells. In addition, in contrast to the L-selectin+ alpha 6-integrinlow phenotype of naive lymph node T cells, IC T cells lacked L-selectin and were alpha 6-integrin-. Mac-1, recently proposed as another marker of memory T cell differentiation, was not displayed by IC T cells, suggesting that Mac-1 expression may be heterogeneous among memory T cell subsets. A subset of mesenteric lymph node (MLN) T cells, probably representing activated T cells undergoing the naive to memory transition, but not of IC T cells, expressed high levels of alpha 6-, beta 7- and alpha E-integrin. IC and MLN naive T cells expressed comparable levels of alpha 4-integrin, but IC T cells stain poorly with anti-beta 7 mAbs and with mAb DATK 32, specific for the alpha 4 beta 7 heterodimeric lymphocyte homing receptor for the mucosal addressin MAdCAM-1, suggesting that these inflammatory cells express more alpha 4 beta 1 than alpha 4 beta 7. Consistent with this, in in vitro adhesion assays, brain IC bound better than MLN cells to the alpha 4 beta 1 integrin ligand VCAM-1 and the LFA-1 ligand ICAM-1 but adhered very poorly to the alpha 4 beta 7 ligand MAdCAM-1. These findings are consistent with and extend previous immunohistological studies of T cells in murine experimental autoimmune encephalomyelitis, and demonstrate a distinctive phenotype for lymphocytes being present in the chronically inflamed brain.

Animals↗
PubMed1994

Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

Animals↗
PubMed1994

Cell adhesion molecules on vessels during inflammation in the mouse central nervous system.

The expression of endothelial cell adhesion molecules (CAMs) in the central nervous system (CNS) of the mouse was examined during an inflammation induced by intracerebral injection of killed Corynebacterium parvum (C. parvum). We showed that injection of killed C. parvum produced an inflammatory cellular infiltrate limited to the injected brain hemisphere. However, the upregulation of ICAM-1 and VCAM-1 on brain endothelium occurred starting 2 days after C. parvum injection throughout the entire CNS and was not restricted to vessels surrounded by a cellular infiltrate. In contrast to the systemic upregulation of ICAM-1 and VCAM-1, cerebral vessels located in the center of the cellular infiltrate started to express the MECA-32 antigen, suggesting an altered functional status of the endothelial cells, as this antigen is suppressed during development of the blood-brain barrier (BBB). Binding assays performed on frozen sections of inflamed brains are consistent with an important role for endothelial VCAM-1 in the recruitment of lymphocytes during inflammation in the CNS of the mouse.

Animals↗
PubMed1994

Lysis of rat brain microvascular endothelial cells mediated by resting but not activated MBP-specific CD4+ T cell lines.

Previous work from this laboratory showed that the encephalitogenic potential of myelin basic protein (MBP)-specific T cells is inseparably associated with their cytotoxic potential. MBP-specific T cells lyse all cells that present autoimmunogenic MBP peptide in context of appropriate MHC class II determinants. Beside class II-induced glia cells, blood-brain barrier-derived endothelial cells were identified as highly susceptible target cells for cytotoxic MBP-specific T cells. Here we show that the cytotoxic reaction against endothelial cells essentially differs from cytotoxicity against other target cells. In contrast to classical T cell-mediated lytic responses, which are most efficiently executed by activated T cells, rat brain endothelium (RBE) lysis could only be mediated by resting T cells. Activated MBP-specific T cell blasts were not able to mediate strong RBE lysis. Furthermore, T cell lines with specificities for protein antigens other than MBP did not cause RBE lysis. A role of the cytolytic capacity of resting MBP-specific T cells in the pathogenesis of experimental autoimmune encephalomyelitis is probable.

Animals↗
PubMed1994

Serum vitamin A levels in respiratory syncytial virus infection.

Respiratory syncytial virus causes worldwide epidemics of respiratory disease. Of 23 children infected with respiratory syncytial virus, 65% had low serum concentrations of vitamin A during acute illness; these low values were associated with more severe illness. Vitamin A supplementation may have a role in the management of infection with respiratory syncytial virus.

Case-Control Studies↗
PubMed1994

Evidence for involvement of ICAM-1 and VCAM-1 in lymphocyte interaction with endothelium in experimental autoimmune encephalomyelitis in the central nervous system in the SJL/J mouse.

We have investigated the expression of vascular adhesion molecules during the first stage of chronic inflammation in experimental autoimmune encephalomyelitis in the SJL/J mouse. Immunocytochemical analysis of frozen sections of inflamed versus noninflamed brains and spinal cords showed that the vascular endothelium in brains and spinal cords from diseased animals expressed high levels of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) but no detectable mucosal addressin or peripheral lymph node addressin. In frozen section assays, anti-alpha 4 integrin and anti-VCAM-1 monoclonal antibodies inhibited binding of mouse peripheral lymphocytes to inflamed brains at both 4 C and 20 C. Antilymphocyte function-associated antigen-1 and anti-ICAM-1 monoclonal antibodies inhibited binding of mouse peripheral lymphocytes to inflamed brains at 20 C. These results are consistent with an important role for the vascular adhesion molecules VCAM-1 and ICAM-1 and for their lymphocytes receptors in lymphocyte recruitment to the central nervous system.

Animals↗
PubMed1992

Induction of persistently demyelinated lesions in the rat following the repeated adoptive transfer of encephalitogenic T cells and demyelinating antibody.

A chronic relapsing model of demyelinating experimental allergic encephalomyelitis (EAE) was induced in Lewis rats by the repeated co-transfer of encephalitogenic, myelin basic protein (MBP)-specific T cells in combination with a demyelinating monoclonal antibody (mAb) specific for the myelin oligodendrocyte glycoprotein (MOG). In controls, repeated injections of 5 x 10(5) MBP-specific T cells at intervals of 18-21 days resulted in an increasing resistance to the induction of further episodes of EAE. However, intravenous injection of the mAb 4 days after each T cell transfer overcame this 'vaccination' effect and induced severe clinical relapses associated with an increasing and persistent neurological deficit. Histological examination revealed that four cycles of treatment with T cells and mAb were sufficient to result in the formation of large plaques of demyelination in the spinal cord that failed to undergo significant remyelination within 60 days of the final injection of mAb. These lesions consisted of a matrix of astrocytic scar tissue traversed by numerous naked axons. These observations demonstrate that the formation of large, persistently, demyelinated lesions in a T cell-mediated model of EAE in the Lewis rat is dependent on the presence of an appropriate anti-myelin autoantibody response.

Animals↗
PubMed1991

Interaction of T lymphocytes with cerebral endothelial cells in vitro.

As a prerequisite of inflammatory lesion formation in (auto-)immune disease of the central nervous system, lymphocytes have to interact with brain endothelia. In recent years much progress has been made towards a better understanding of mechanisms and factors involved in organ specific homing of lymphocytes. Many lines of evidence indicate that T lymphocytes recognizing antigens which are exclusively beyond the blood-brain barrier cross this barrier only when they are in an activated state, irrespective of their antigen specificity. Antigen presentation by blood-brain barrier endothelia, however, may play a role in later stages of florid inflammation.

Animals↗
PubMed1990

Double-blind, placebo-controlled trial of alternate-day furosemide therapy in infants with chronic bronchopulmonary dysplasia.

To test the hypothesis that alternate-day administration of furosemide will result in a sustained improvement in pulmonary function without causing alterations in electrolyte or mineral homeostasis, we conducted a randomized, double-blind, placebo-controlled study of 11 hospitalized, oxygen-dependent, spontaneously breathing infants with chronic bronchopulmonary dysplasia. Infants were randomly selected to receive either furosemide, 4 mg/kg in two divided doses on alternate days orally, or placebo for 8 days, followed by crossover to the alternate-therapy for an additional 8-day period. The two study periods were separated by a 48-hour washout period. Dynamic compliance, total pulmonary resistance, the concentration of electrolytes in serum, and the concentrations of calcium and creatinine in urine were measured on nontreatment days. Alternate-day furosemide therapy increased dynamic lung compliance by 76 +/- 112% and decreased total pulmonary resistance by 20 +/- 39%, compared with placebo (both variables p = 0.032). Alternate-day furosemide therapy did not result in increased urine output, electrolyte abnormalities, or increased urinary calcium excretion. We conclude that this simplified treatment regimen may be useful in the management of infants with chronic bronchopulmonary dysplasia. The results support our previous speculation that furosemide improves pulmonary function by mechanisms unrelated to its diuretic properties.

Airway Resistance↗
PubMed1990

Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro.

The endothelial blood-brain barrier (BBB) has a critical role in controlling lymphocyte traffic into the central nervous system (CNS), both in physiological immunosurveillance, and in its pathological aberrations. The intercellular signals that possibly could induce lymphocytes to cross the BBB include immunogenic presentation of protein (auto-)antigens by BBB endothelia to circulating T lymphocytes. This concept has raised much, though controversial, attention. We approached this problem by analyzing in vitro immunospecific interactions between clonal rat T lymphocyte lines with syngeneic, stringently purified endothelial monolayer cultures from adult brain micro-vessels. The rat brain endothelia (RBE) were established from rat brain capillaries using double collagenase digestion, density gradient fractionation and selective cytolysis of contaminating pericytes by anti-Thy 1.1 antibodies and complement. Incubation with interferon-gamma in most of the brain-derived endothelial cells induced Ia-antigens in the cytoplasm and on the cell surface in some of the cells. Before the treatment, the cells were completely Ia-negative. Pericytes were unresponsive to IFN-gamma treatment. When confronted with syngeneic T cell lines specific for protein (auto-)antigens (e.g., ovalbumin and myelin basic protein, MBP), RBE were completely unable to induce antigen-specific proliferation of syngeneic T lymphocytes irrespective of pretreatment with IFN-gamma and of cell density. RBE were inert towards the T cells, and did not suppress T cell activation induced by other "professional" antigen presenting cells (APC) such as thymus-derived dendritic cells or macrophages. IFN-gamma-treated RBE were, however, susceptible to immunospecific T cell killing. They were lysed by MBP-specific T cells in the presence of the specific antigen or Con A. Antigen dependent lysis was restricted by the appropriate (MHC) class II product. We conclude that the interaction of brain endothelial cells with encephalitogenic T lymphocytes may involve recognition of antigen in the molecular context of relevant MHC products, but that this interaction per se is insufficient to initiate the full T cell activation program.

Animals↗
PubMed1989

Immunotherapy of experimental autoimmune encephalomyelitis (EAE): differential effect of anti-IL-2 receptor antibody therapy on actively induced and T-line mediated EAE of the Lewis rat.

Treatment of Lewis rats with monoclonal anti-interleukin-2 receptor (IL-2 R) antibody ART-18 is highly efficient in protecting the recipients from T-line transferred experimental autoimmune encephalomyelitis (tEAE) in vivo. In contrast, ART-18 did not affect the development of EAE actively induced (aEAE) by immunization with myelin basic protein (MBP) in complete Freund's adjuvant (CFA). ART-18 caused a slight delay in the development of aEAE only in combination with a subtherapeutic dose of cyclosporine A (Cy-A), but failed to influence duration or severity of clinical signs. The discrepancy in therapeutic efficiency of ART-18 in tEAE and aEAE could be due to a different intensity of IL-2 R-expression on in vitro- and in vivo-activated MBP-specific T cells. Our results therefore caution against a general therapeutic application of anti IL-2 R-directed therapy in all manifestations of T-cell-mediated autoimmunity.

Animals↗
PubMed1989

Effect of spironolactone-hydrochlorothiazide on lung function in infants with chronic bronchopulmonary dysplasia.

To test the hypothesis that spironolactone-hydrochlorothiazide (Aldactazide) will improve urine output and lung function in infants with bronchopulmonary dysplasia, we studied 21 hospitalized, spontaneously breathing, oxygen-dependent infants with chronic bronchopulmonary dysplasia. Infants were randomly assigned to receive either a 1:1 mixture of spironolactone and hydrochlorothiazide orally (n = 12) (3 mg/kg/day of both compounds) or no treatment (n = 9) for 6 to 8 days each. Dynamic lung compliance, total pulmonary resistance, and hemoglobin oxygen saturation were measured on the first and last days of each study period. Fluid intake and urine output were measured each day. Although the treatment significantly increased urine output, neither lung mechanics nor oxygenation were improved by the drug. The magnitude of the diuresis achieved with spironolactone-hydrochlorothiazide treatment was comparable to the diuresis achieved in a previous study of furosemide treatment (J Pediatr 1986:109;1034-9). Statistical analysis indicated that a type II error was an unlikely explanation for our failure to detect a beneficial effect. In three patients, doubling the oral dose did not improve lung mechanics or oxygenation. We speculate that diuresis per se is not responsible for lung function improvement during treatment with other drugs with diuretic properties.

Bronchopulmonary Dysplasia↗