Chemokines have been convincingly implicated in actuating inflammatory leukocyte emigration. To affect the circulating leukocytes, tissue-derived chemokines have to traverse the endothelial cells (ECs). This was thought to be accomplished by chemokine diffusion through the intercellular gaps. On the contrary, we show by electron microscopy that the prototype chemokine IL-8 is internalized by venular ECs abluminally and transcytosed to the luminal surface. Here, it is presented to the adherent leukocytes on the EC membrane, predominantly in association with the EC projections. The intact C terminus of IL-8, the molecule's "immobilization" domain, is required for the EC binding, transcytosis, and consequently, the in vivo proemigratory activity of IL-8, indicating that the described subcellular interactions of IL-8 with the ECs are functionally relevant.

SummaryThe expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC.The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in WeibelPalade bodies may serve as the EC "memory" of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.

Chemokines have been convincingly implicated in driving leukocyte emigration in different inflammatory reactions. However, the cellular and molecular mechanisms of chemokine involvement in leukocyte emigration are not clear. We and others suggested that leukocyte adhesion to the endothelium and transmigration are induced by chemokines immobilized on the endothelial cell surface. This would require the presence of specific chemokine binding sites in this microanatomical location. Using an in situ binding assay we demonstrated the presence of binding sites for interleukin-8 (IL-8) and RANTES, but not monocyte inflammatory protein-1 alpha on the endothelium of postcapillary venules and small veins in human skin. In contrast, venules and veins in various anatomical locations showed dramatically differing IL-8 binding patterns. The subcellular distribution of IL-8 in the venular endothelial cells following its in vivo and ex vivo injections was studied by use of electron microscopy. Our results suggest that IL-8 was internalized by the endothelial cells, transported transcellularly via plasmalemmal vesicles, and released onto the luminal surface where it appeared located preferentially on tips of membrane protrusions. We were unable to study the endothelial IL-8 binding or transport in vitro because all the in vitro propagated endothelial cell lines and primary endothelial cells tested lacked IL-8 binding sites. This includes human umbilical vein endothelial cells (HUVECs), which also did not bind IL-8 in situ. However, HUVECs provided a satisfactory in vitro system to study the secretion of IL-8 by the endothelial cells. Two possible alternative pathways were described: secretion directly from the Golgi apparatus or following storage in Weibel-Palade bodies.

Insulin-like growth factor-I (IGF-I) stimulates the production of extracellular matrix by cartilage cells and this action is mediated through the Type 1 IGF receptor. Expression of the genes for the IGF receptor and for IGF-I was examined in normal and osteoarthritic (OA) human articular cartilage by in situ hybridization. RNA transcripts for Type 1 receptor were detected in all 73 tissue samples and in 80-100% of chondrocytes per section. Signal for the receptor was present in normal and OA cells, and the highest message levels were in the tissues exhibiting advanced pathology. Strong message signals in the ceUs of the more advanced lesionS were also noted for IGF-I, whereas little or no IGF-I mRNA was IntroductionArticular cartilage consists of chondrocytes embedded in an extensive extracellular matrix. These cells synthesize, organize, and regulate the deposition of their surrounding matrix, and in normal mature tissue they actively maintain a stable equilibrium between synthesis and degradation of matrix molecules. In disease states such as osteoarthritis (OA), the stable equilibrium is disrupted, leading eventually to complete loss of cartilage from the joint surface. Reports of altered phenotypic expression in human OA chondrocytes include production of Type 111 and Type X collagen (I), the detection of novel chondroitin sulfate epitopes attached to aggrecan (2), progressive loss of extracellular matrix, and the formation of clonal cell clusters in depleted regions (3).Insulin-like growth factors (IGFs) or somatomedins regulate the growth and differentiation of a variety of tissues and are implicated in their hypertrophy and repair (4). They are bound in vivo to specific binding proteins (IGF BP 1-6) which extend the half-life of the growth factor and modulate its activity (5). Traditionally, IGF-I Supported by the Nuffield Foundation (Oliyr Bird Fund) and by the Arthritis and Rheumatism Council.Correspondence tu James Middleton, PhD, Dept. of General Dermatology, Sandot Forschungsinstitut, Brunnerstr. 59, A-1235 Vienna, Austria. was considered to be mainly produced in the liver and to mediate the actions of growth hormone on target tissues (endocrine function). However, it is now clear that IGFI is synthesized by many cells of mesenchymal origin in different tissues, indicating a local function involving autocrinelparacrine mechanisms. IGFI is a major anabolic growth factor in the regulation of articular cartilage metabolism in vitro (6,7). It also stimulates the growth of epiphyseal cartilage in vivo (8). Rat epiphysial chondrocytes express the IGF-I gene, and locally produced IGFI is implicated in stimulating clonal expansion of these cells (9). In a previous study we showed that chondrocytes in human articular cartilage also express IGFI mRNA. Very low amounts were detected in normal samples, whereas enhanced levels, four-to fivefold higher, were evident in cells from OA cartilage that had formed clusters at fibrillated sites (10). The metabolic and mitogenic action of IGFs are believed to be mediated...

Expression of insulin-like growth factor I (IGF-I) mRNA and IGF-I protein was studied in human osteoarthritic and young articular cartilage by in situ hybridisation and immunohistochemistry. In situ hybridisation showed that relatively low amounts of IGF-I mRNA signal were present in anatomicaliy normal regions of osteoarthritic and young cartilage.In fibrillated osteoarthritic cartilage, however, the signal intensity was significantly higher than in non-fibrillated cartilage. Particularly high levels of IGF-I mRNA were present in the surface cell clones of more advanced lesions, the amount of signal being about four to five times greater than in anatomically normal cartilage. The amount of message varied with cartilage depth. In young cartilage there was less IGF-I mRNA in the superficial zone than in the middle and deep zones. In fibrillated regions of osteoarthritic joints the amount of message in surface cells was greater than in deeper regions. A specific human IGF-I antibody was used to show the presence intracellularly of IGF-I protein in osteoarthritic and young cartilage.Raised levels of IGF-I message in osteoarthritic chondrocytes may represent an attempt at increased matrix repair, operating by an autocrine/paracrine mechanism.

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The schizont stage of the protozoan parasite Theileria parva induces features characteristic of tumor cells in infected bovine T-cell lines. Most strikingly T. parvainfected cell lines acquire unlimited growth potential in vitro. Their proliferative state is entirely dependent on the presence of a viable parasite within the host cell cytoplasm. It has been postulated that parasite proteins either secreted into the host cell or expressed on the parasite surface membrane are involved in the parasitehost cell interaction. We used an in vitro transcriptiontranslation-membrane translocation system to identify T. parva-derived cDNA clones encoding secretory or membrane proteins. Within 600 clones we found one encoding a 17-kDa protein which is processed by microsomal membranes to a 14-kDa protein (11E), presumably by signal peptidase. The processed form is expressed in the T-cell line TpM803 harboring viable parasites. By immunolocalization we show that the 11E protein mostly resides within the parasite, often in close vicinity to membranous structures, but in addition it appears at the surface membrane. Amino acid sequence comparison suggests that 11E belongs to the glutaredoxin family, but is unique so far in containing a signal sequence for endoplasmic reticulum membrane translocation.

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