Elke Brylla

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

Microvascular Endothelial Cells Differ in Their Basal and Tumour Necrosis Factor-α-Regulated Expression of Adhesion Molecules and Cytokines

We recently located a rare cytokeratin-positive (CK+) type of microvascular endothelial cell (MVEC) in the corpus luteum and aorta. Bovine corpus luteum MVEC are known to be involved in the cyclic accumulation of eosinophils and macrophages. Since leukocyte migration is specifically mediated by adhesion molecules and the release of cytokines, we compared the expression of these factors in basal and TNF-α-stimulated CK+ MVEC and in common cytokeratin-negative (CK–) MVEC in order to obtain an initial insight into the functional capacities of CK+ MVEC. CK– MVEC revealed significantly higher basal RANTES mRNA expression than CK+ MVEC, and TNF- α up-regulated RANTES mRNA in both types of MVEC. Only resting and stimulated CK– MVEC expressed granulocyte-macrophage colony-stimulating factor mRNA. Both MVEC types expressed monocyte colony-stimulating factor mRNA, but remained negative for eotaxin and interleukin (IL)-5 mRNA even after stimulation. Resting CK+ MVEC were positive for CD29, CD31, CD49a and CD49e, but expressed most of these antigens at a significantly lower density than did CK– MVEC. In contrast to CK– MVEC, CK+ MVEC failed to express CD49b or MHC class II. The activation of CK+ MVEC with TNF-α induced the expression of CD62P, but not of CD49b or MHC class II. In summary, phenotypically variable MVEC derived from the microvascular bed of one organ differ in their TNF-α-regulated expression of cytokine mRNA and adhesion molecules. Morphological heterogeneity is related to a particular specialisation of functional MVEC.

IL-6 Regulates Neutrophil Microabscess Formation in IL-17A-Driven Psoriasiform Lesions

The lack of a generally accepted animal model for human psoriasis has hindered progress with respect to understanding the pathogenesis of the disease. Here we present a model in which transgenic IL-17A expression is targeted to the skin in mice, achievable after crossing our IL-17A(ind) allele to the K14-Cre strain. K14-IL-17A(ind/+) mice invariably develop an overt skin inflammation bearing many hallmark characteristics of human psoriasis including dermal infiltration of effector T cells, formation of neutrophil microabscesses, and hyperkeratosis. IL-17A expression in the skin results in upregulated granulopoiesis and migration of IL-6R-expressing neutrophils into the skin. Neutralization of IL-6 signaling efficiently reduces the observed pathogenesis in skin of IL-17A-overexpressing mice, with marked reductions in epidermal neutrophil abscess formation and epidermal thickening. Thus, IL-6 functions downstream of IL-17A to exacerbate neutrophil microabscess development in psoriasiform lesions.

Coexpression of preprotachykinin A and B transcripts in the bovine corpus luteum and evidence for functional neurokinin receptor activity in luteal endothelial cells and ovarian macrophages

Nonneuronal cell sources of tachykinins, such as substance P (SP) and neurokinin B (NKB), have been demonstrated in leukocytes, endothelial cells and endocrine cells, and may play a role in corpus luteum (CL) development. For this reason, we analyzed mRNA presence for the two tachykinin precursors together with the neurokinin-1 receptor and the neurokinin-3 receptor (NK-1R and NK-3R, preferred by SP and NKB, respectively) in bovine CL at various stages in the luteal phase. Using the RT-PCR technique, we detected coexpression for the preprotachykinin A gene (PPT-A), which encodes SP and neurokinin A (NKA), and the preprotachykinin B gene (PPT-B) for NKB in the CL at the development, secretion and regression stages. Coexpression was also noted for NK-1R and NK-3R gene transcripts. Cultures of endothelial cells (ECs) derived from bovine CL expressed NK-1R and NK-3R mRNA, as did ovarian macrophages. Agonist treatment induced a stronger intracellular calcium ([Ca2+]i) increase after activation of NK-1R compared to NK-3R, a result that we verified by calcium imaging. This is the first evidence for functional tachykinin receptor activity in luteal ECs and ovarian macrophages from bovine CL.

Differences between retinal and choroidal microvascular endothelial cells (MVECs) under normal and hypoxic conditions.

The morphological and functional differences between the retinal and choroidal vascular bed raise the question of whether the smallest functional unit, the microvascular endothelial cell (MVEC), also differs in its basal characteristics. Here, we examined bovine retinal and choroidal MVECs (rMVECs, cMVECs) for the presence and regulation of angiogenic mediators and their receptors, and cytokines at the mRNA level using quantitative RT-PCR and differential display. Vascular endothelial growth factor (VEGF) mRNA was expressed in both rMVECs and cMVECs. The basal and hypoxia-increased VEGF mRNA levels were significantly higher in cMVECs, which may indicate a higher capacity for autocrine stimulation in these cells. The mRNA for two VEGF receptors, Flt-1 and Flk-1, was present in rMVECs and cMVECs. Interestingly, rMVECs expressed higher Flt-1 but lower Flk-1 mRNA levels compared to cMVECs. Examining the angiopoietin (Ang)/Tie-2 system, we only detected Ang-1 mRNA at very low levels. While Ang-2 mRNA levels were high in both rMVECs and cMVECs, rMVECs expressed 2-3 times the basal and hypoxia-upregulated Ang-2 mRNA levels than did cMVECs. No difference was found in basal Tie-2 mRNA levels. rMVECs are the more potent producers of macrophage colony-stimulating factor (M-CSF) and granulocyte-macrophage CSF (GM-CSF), whereas cMVECs expressed higher RANTES mRNA levels. In our second approach - screening rMVECs and cMVECs for differentially expressed genes - we found liprin-beta1, calnexin, and sushi-repeat-containing protein, x chromosome (SRPX) mRNA in both MVEC types at varying levels. In summary, MVECs from the retinal and choroidal vascular beds showed quantitative differences in angiogenic regulator expression and in their capability to produce cytokines.

Cloning of bovine RANTES mRNA and its expression and regulation in ovaries in the periovulatory period.

RANTES may be one of the chemoattractants involved in stimulating eosinophils and macrophages to migrate selectively into bovine dominant follicles and into developing corpora lutea. We sequenced a 736 bp fragment of the bovine RANTES mRNA encoding the complete protein and defined the ovarian source of RANTES mRNA. As demonstrated by competitive RT‐PCR, follicle‐derived macrophages showed a 100–1000 times higher RANTES mRNA level compared to unpurified granulosa cells or follicle‐derived fibroblasts. By means of in situ hybridization, RANTES mRNA positive macrophages were located in the former thecal layer of the developing corpora lutea.

KIT receptor-positive cells in the bovine corpus luteum are primarily theca-derived small luteal cells

The tyrosine kinase KIT receptor, the protooncogene CD117, plays a key role in growth and maturation of oocytes and follicles. Relevant data are sparse for the corpus luteum (CL). We first confirmed the presence of KIT mRNA and KIT protein in bovine CL homogenates. We then localized KIT-positive (KIT+) cells in CL sections by immunohistochemistry. At the CL stage of early development, the former theca transforming into capsule/septa showed a strong band-like KIT+ immunoresponse. In addition, CD45+ leukocytes in septa included subpopulations of CD45+/KIT+ and CD14+/KIT+ leukocytes as validated by double immunofluorescence localization. At the early secretory stage, KIT+ cells appeared within the septa/capsule region and in the periphery of the CL parenchyma, there forming a complex network. This was separate from the capillary bed as determined by double staining for CD117 and FVIII-related endothelial cell antigen (FVIIIr). The KIT+ network coincided with cells positive for cytochrome P450 17alpha-hydroxylase, a thecal cell-specific enzyme. The late secretory stage was defined by an advanced manifestation of the KIT+ network in the CL periphery. At the stage of regression, the KIT+ network was absent. The CL of pregnancy expressed high levels of KIT mRNA and KIT protein uniformly throughout pregnancy. The KIT+ immunolocalization revealed small fibroblast-like cells, luteal cells with granules, and clusters of large luteal cells with staining of the cell membrane. We conclude that a majority of KIT+ cells in the bovine CL are primarily theca-derived small luteal cells, and that a minority represent KIT+ leukocytes, in some cases KIT+ monocytes.

Transcripts of neurokinin B and neurokinin 3 receptor in superovulated rat ovaries and increased number of corpora lutea as a non-specific effect of intraperitoneal agonist application.

Neurokinin B (NKB), a member of the tachykinin family, and its neurokinin 3 receptor (NK3-R) are preferentially found in the central nervous system. Others have recently reported on mRNA from this ligand-receptor system in the uterus and on NK3-R expression increasing with age. NKB and NK3-R mRNAs have also been noted in cumulus cells and oocytes from superovulated rats. Intact ovaries before and after puberty have not been studied. In this study, we stimulated 29-day-old rats by s.c. injections with gonadotropins for estrous cycle synchronization in order to elucidate the NKB-NK3-R systems expression and function in the ovary. Simultaneously, NaCl, the NK3-R agonist (Pro(7))-NKB, the antagonist SB 218795, or thiorphan, a neutral endopeptidase inhibitor of tachykinin degradation, were injected intraperitoneally (i.p.) for 3 1/2 consecutive days. First, we demonstrated NKB and NK3-R transcripts in one rat ovary by RT-PCR. No significant mRNA differences were noted between immature ovaries and superovulated ovaries in any of the i.p. applications. Second, the possible role of NK3-R on the ovulatory process was verified by counting corpora lutea (CL) and CL cysts in serial sections of the other ovary derived from the four different groups and embedded in paraffin wax. CL and CL cysts were noted in greater numbers in the pharmacologically treated groups than in the saline-treated group. To validate possible drug effects on the peritoneum, we additionally studied pieces of the omentum majus and retroperitoneal fat tissue. Both tissues were heavily infiltrated by granulocytes similar to a non-specific inflammatory response. The saline-treated group as well as the pharmacologically treated groups appeared to develop this unexpected side effect to a similar degree. We conclude that transcripts of NKB and NK3-R are present before and after puberty in the rat ovary and appear to be expressed at similar levels which may indicate a role for the NKB-NK3-R system in follicle growth. The effect of increased CL formation after application of the NK3-R agonist i.p. is related to a non-specific response.

Identification of Inwardly Rectifying Potassium Channels in Bovine Retinal and Choroidal Endothelial Cells

Ion channels were studied using the whole-cell patch clamp technique in bovine retinal and choroidal microvascular endothelial cells (MVEC) cultured under the same conditions. The two types of MVEC expressed inward currents at hyperpolarizing voltage steps and showed small outward currents at depolarizing steps. The extrapolated reversal potentials of the inward currents were near to the potassium equilibrium potential. Cs+ and the K+ channel blocker TEA reduced the amplitudes of the currents indicating the selectivity and permeability for potassium. This was confirmed by changes of outside K+ concentration shifting the I-V curves to the right. RT-PCR studies revealed the presence of mRNA of Kir2.1, an inwardly rectifying K+ channel, in retinal and choroidal MVEC. The profile of the small outward currents is related to the Kv family but not identical with the Kv1.4 subtype.

Cryopreserved Porcine Tendons Preserve Cell Viability After Thawing

Controlled cryopreservation is an important method for storage of tissue grafts in skin banking, reproductive medicine and other domains. Although the availability of cryopreserved flexor tendons would be highly beneficial in reconstructive surgery, especially in complex reconstructions for which grafting material is limited, only a few studies have dealt with transplanted tendons. We achieved successful cryopreservation of porcine flexor tendons in 2 cryoprotective media: dimethyl sulfoxide and glycerol. Their viability was shown using a quantitative colorimetric MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. For comparison of native and cryopreserved tendons (n = 7 samples each), the adopted viability index was the ratio of MTT-dependent optical density and tendon weight. The viability index of native samples did not change significantly after cryopreservation and thawing. The proliferative capacity of tendon fibroblasts after thawing was shown in primary cell cultures. The described cryopreservation protocol and MTT assay may provide a basis for future autografting of human tendons.