Rupert Hallmann

Differentiation-dependent expression of proteins in brain endothelium during development of the blood-brain barrier

The blood-brain barrier is a specific property of differentiated brain endothelium. To study the differentiation of blood vessels in the brain, we have correlated the expression of a number of proteins in brain endothelial cells with the development of the blood-brain barrier in mouse, quail, and chick embryos. Using histochemical methods, alkaline phosphatase activity was found to be present in all species and appeared around embryonic Days 17 (mouse), 14 (quail), and 12 (chick). Butyrylcholinesterase activity was found in the mouse and quail but not the chick brain vasculature, and appeared around Days 17 (mouse) and 15 (quail). gamma-Glutamyltranspeptidase activity was demonstrated histochemically in mouse but not in chick and quail brain capillaries, beginning at Day 15. Transferrin receptor was localized on brain endothelium in all species by immunofluorescence methods using monoclonal antibodies. It appeared at Days 15 and 11 in mouse and chick embryonic brain, respectively. The staining of all markers in embryonic brain was compared with adult brain endothelium and the leptomeningeal blood vessels. The expression of these proteins was correlated with the development of the blood-brain barrier by studying the permeability of brain endothelium for the protein horseradish peroxidase during mouse embryogenesis. Vessels in the telencephalon were found to become impermeable around Day 16 of development. Taken together the results of previous investigations and those presented here, we conclude that a number of proteins are sequentially expressed in brain endothelial cells correlating in time with the formation of the blood-brain barrier in different species.

Endothelial basement membrane laminin |[alpha]|5 selectively inhibits T lymphocyte extravasation into the brain

Specific inhibition of the entry of encephalitogenic T lymphocytes into the central nervous system in multiple sclerosis would provide a means of inhibiting disease without compromising innate immune responses. We show here that targeting lymphocyte interactions with endothelial basement membrane laminins provides such a possibility. In mouse experimental autoimmune encephalomyelitis, T lymphocyte extravasation correlates with sites expressing laminin α4 and small amounts of laminin α5. In mice lacking laminin α4, laminin α5 is ubiquitously expressed along the vascular tree, resulting in marked and selective reduction of T lymphocyte infiltration into the brain and reduced disease susceptibility and severity. Vessel phenotype and immune response were not affected in these mice. Rather, laminin α5 directly inhibited integrin α6β1–mediated migration of T lymphocytes through laminin α4. The data indicate that T lymphocytes use mechanisms distinct from other immune cells to penetrate the endothelial basement membrane barrier, permitting specific targeting of this immune cell population.

Brain induces the expression of an early cell surface marker for blood-brain barrier-specific endothelium.

Capillaries derived from the perineural vascular plexus invade brain tissue early in embryonic development. Considerably later they differentiate into blood‐brain barrier (BBB)‐forming blood vessels. In the chick, the BBB as defined by impermeability for the protein horseradish peroxidase develops around embryonic day 13. We have previously found that brain endothelial cells start to express a number of proteins at around the same time, suggesting that these proteins play a role in BBB function. Here we describe a 74 kd protein defined by the monoclonal antibody HT7 that is expressed on the surface of chick embryonic blood cells and brain endothelial but on no other endothelial cells. This protein is not detectable on early embryonic brain endothelium, but is expressed by these cells on embryonic day 10. It is absent in choroid plexus endothelial cells which represent permeable fenestrated endothelial cells. The antigen is expressed on choroid plexus epithelium which is the site of the blood‐cerebrospinal fluid barrier. Since it is also found in basolateral membranes of kidney tubules, it may be involved in specific carrier mechanisms. Embryonic mouse brain tissue transplanted on the chick chorio‐allantoic membrane induces the expression of this antigen on endothelial cells derived from the chorio‐allantois. Brain tissue can therefore induce in endothelial cells in vivo the expression of a molecule characteristic of brain endothelium.

TNF-α-Induced Expression of Adhesion Molecules in the Liver Is Under the Control of TNFR1—Relevance for Concanavalin A-Induced Hepatitis

TNF-α has been clearly identified as central mediator of T cell activation-induced acute hepatic injury in mice, e.g., Con A hepatitis. In this model, liver injury depends on both TNFRs, i.e., the 55-kDa TNFR1 as well as the 75-kDa TNFR2. We show in this report that the hepatic TNFRs are not transcriptionally regulated, but are regulated by receptor shedding. TNF directly mediates hepatocellular death by activation of TNFR1 but also induces the expression of inflammatory proteins, such as cytokines and adhesion molecules. Here we provide evidence that resistance of TNFR1−/− and TNFR2−/− mice against Con A hepatitis is not due to an impaired production of the central mediators TNF and IFN-γ. Con A injection results in a massive induction of ICAM-1, VCAM-1, and E-selectin in the liver. Lack of either one of both TNFRs did not change adhesion molecule expression in the livers of Con A-treated mice, presumably reflecting the fact that other endothelial cell-activating cytokines up-regulated adhesion molecule expression. However, treatment of TNFR1−/− and TNFR2−/− mice with murine rTNF revealed a predominant role for TNFR1 for the induction of hepatic adhesion molecule expression. Pretreatment with blocking Abs against E- and P-selectin or of ICAM−/− mice with anti-VCAM-1 Abs failed to prevent Con A hepatitis, although accumulation of the critical cell population, i.e., CD4+ T cells was significantly inhibited. Hence, up-regulation of adhesion molecules during acute hepatitis unlikely contributes to organ injury but rather represents a defense mechanism.

Low-dose radiotherapy selectively reduces adhesion of peripheral blood mononuclear cells to endothelium in vitro☆

BACKGROUND AND PURPOSE The anti-inflammatory effect of low-dose radiotherapy (LD-RT) still is not understood. The adhesion of leukocytes to endothelial cells (EC) of the vessel wall is the initial event of tissue invasion, and thus, crucially contributes to the regulation of inflammation. We investigated the influence of LD-RT on the adhesion process in vitro. MATERIALS AND METHODS Isolated peripheral-blood-mononuclear-cells (PBMC) were incubated with an activated murine endothelioma cell-line under shear conditions at 4 degrees C after irradiation with single doses between 0.1 and 10.0 Gy. Adherent cells were counted microscopically and compared to a non-irradiated control. In parallel, viability and expression of adhesion molecules, especially of L-selectin, and lineage-specific markers on the cell surface were determined by dye exclusion and cytofluorometry, respectively. Modulation of adhesion by soluble L-selectin was tested in the adhesion assay. RESULTS Radiation doses of 0.1-0.5 Gy reduced the adhesion of viable PBMC to EC in vitro by 70% of the control level 4 h after irradiation. Leukocytes showed a marked reduction of L-selectin expression after LD-RT. Soluble L-selectin can inhibit the adhesion of PBMC to EC. CONCLUSION The anti-inflammatory effect of LD-RT might, in part, be due to the reduction in the adhesion of PBMC to EC. This reduction in adhesion might be a consequence of the reduced expression of L-selectin on the surface of PBMC, and the inhibition of adherence by soluble L-selectin shed by PBMC in vitro.

Alterations in the Expression of Homing-Associated Molecules at the Maternal/Fetal Interface During the Course of Pregnancy

Abstract One of the most fascinating immunologic questions is how the genetically distinct fetus is able to survive and develop within the mother without provoking an immune rejection response. The pregnant uterus undergoes rapid morphological and functional changes, and these changes may influence the nature of local immune responses at the maternal/fetal interface at different stages of gestation. We hypothesized that specialized mechanisms exist to control access of maternal leukocyte subsets to the decidua and that these mechanisms are modulated during the course of pregnancy. At the critical period of initial placenta development, the maternal/fetal interface displays an unparalleled compartmentalization of microenvironmental domains associated with highly differentiated vessels expressing vascular addressins in nonoverlapping patterns and with recruitment of specialized leukocyte subsets (monocytes, granulated metrial gland cells, and granulocytes) thought to support, modulate, and regulate trophoblast invasion. One of the most striking observations at this time of gestation is the almost complete exclusion of lymphocytes from the maternal/fetal interface. The second half of pregnancy is characterized by a partial loss of microenvironmental specialization and different switches in vascular specificity within the decidua basalis, paralleling dramatic changes in the populations of recruited leukocytes (e.g., a striking influx of lymphocytes, especially T cells). In the term pregnant uterus, the expression of all vascular addressins decreased dramatically; only weakly staining maternal vascular segments remained. These segments may define sites of extremely low residual traffic in the term decidua, which contains remarkably few maternal leukocytes overall. Our results suggest that the maternal/fetal interface represents a situation in which leukocyte trafficking is exquisitely regulated to allow entry of specialized leukocyte subsets that may play a fundamental role in immune regulation during pregnancy.

Evidence of specialized leukocyte-vascular homing interactions at the maternal/fetal interface.

In normal pregnancy, the maternal immune system fails to reject the fetus or the placenta as an allogeneic graft. We hypothesize that specialized mechanisms of leukocyte recruitment might limit access of circulating maternal immune cells to the maternal / fetal interface. During the critical period of initial trophoblast invasion there is an elegantly orchestrated progression of leukocyte homing events in the decidua basalis, associated with highly regulated expression of vascular addressins and segregation of specialized leukocyte subsets into well‐defined decidual microdomains. Neutrophils are limited to the region of necrosis associated with enzymatic digestion at the leading edge of the invading trophoblast, where an almost linear array of maternal blood vessels displays the neutrophil ligand E‐selectin. Cells with the phenotype of monocytes but expressing α4β7 integrin are localized in the blood vessels of the specialized “vascular zone”, which display the unusual combination of P‐selectin (partially associated with platelets) and the α4β7 ligand mucosal vascular addressin‐1 (MAdCAM‐1). Granulated metrial gland cells (α4+β7–, probably α4β1+) constitute a well‐defined cluster positioned in the central decidua basalis around venules prominently expressing the α4β1 ligand VCAM‐1 (but not MAdCAM‐1). T and B lymphocytes are rare. Our results suggest that selective mechanisms for regulating leukocyte access, associated with microdomain specialization within the decidua basalis, may play a fundamental role in immune regulation during the invasive period of placental development.

Focal MMP-2 and MMP-9 activity at the blood-brain barrier promotes chemokine-induced leukocyte migration.

Although chemokines are sufficient for chemotaxis of various cells, increasing evidence exists for their fine-tuning by selective proteolytic processing. Using a model of immune cell chemotaxis into the CNS (experimental autoimmune encephalomyelitis [EAE]) that permits precise localization of immigrating leukocytes at the blood-brain barrier, we show that, whereas chemokines are required for leukocyte migration into the CNS, additional MMP-2/9 activities specifically at the border of the CNS parenchyma strongly enhance this transmigration process. Cytokines derived from infiltrating leukocytes regulate MMP-2/9 activity at the parenchymal border, which in turn promotes astrocyte secretion of chemokines and differentially modulates the activity of different chemokines at the CNS border, thereby promoting leukocyte migration out of the cuff. Hence, cytokines, chemokines, and cytokine-induced MMP-2/9 activity specifically at the inflammatory border collectively act to accelerate leukocyte chemotaxis across the parenchymal border.

The extracellular matrix of the spleen as a potential organizer of immune cell compartments.

Until recently little information was available on the molecular details of the extracellular matrix (ECM) of secondary lymphoid tissues. There is now growing evidence that these ECMs are unique structures, combining characteristics of basement membranes and interstitial or fibrillar matrices, resulting in scaffolds that are strong and highly flexible and, in certain secondary lymphoid compartments, also forming conduit networks for rapid fluid transport. This review will address the structural characteristics of the ECM of the murine spleen and its potential role as an organizer of immune cell compartments, with reference to the lymph node where relevant.

Role of the extracellular matrix in lymphocyte migration

The extracellular matrix (ECM) exists in various biochemical and structural forms that can act either as a barrier to migrating leukocytes, in the case of basement membranes, or provide a physical scaffold supporting or guiding migration (interstitial matrix). This review focuses on basement membranes and our current knowledge of the way that leukocytes transmigrate this protein barrier, with emphasis on T lymphocytes. Recent data suggest that the classical concept of cell-matrix adhesion requires revision with respect to leukocyte-ECM interactions. Whereas specific receptors may be required for leukocyte recognition of ECM molecules or three-dimensional structural domains, the role of adhesion in migration as perceived from the traditional studies of adherent cell-ECM interactions is less clear. Further, the indirect effects of ECM such as the binding and presentation of cytokines or chemotactic factors may more profoundly influence the directed migration of normally non-adherent leukocytes than the migration of adherent cells such as epithelial cells or fibroblasts. Proteases (in particular matrix metalloproteinases) released at sites of inflammation can selectively process ECM, cell surface molecules or soluble factors, which may result in the release of bioactive fragments that can function as chemoattractants for different leukocyte subsets or may modulate the activity/function of resident mesenchymal and immune cells. Current findings suggest that different leukocyte types employ different mechanisms to migrate across or through the ECM; this might be determined by the composition and organization of the ECM itself.