No abstract

We report here that interleukins have a dramatic effect on extracellular matrix production by cultured endothelial cells. Human umbilical vein endothelial cells incubated with growth media conditioned by lectin-activated human peripheral blood mononuclear leukocytes undergo marked changes in cell shape and elaborate a highly organized extracellular material that is not detectable in untreated cultures. This material has the following characteristics: (a) it is not recognizable by electron microscopy unless the cationic dye, Alcian blue, is added to the fixative; (b) it is visualized as a network of branching and anastomosing fibrils of various thickness that can be resolved into bundles of fine filaments; (c) it is associated with the cell surface, extends between contiguous cells, and coats the culture substrate; (d) it is removed by digestion with glycosaminoglycan-degrading enzymes, such as crude heparinase and chondroitinase ABC. These results demonstrate that soluble factors released by activated peripheral blood mononuclear leukocytes (interleukins) stimulate cultured human umbilical vein endothelial cells to produce a highly structured pericellular matrix containing glycosaminoglycans (probably chondroitin sulfate and/or hyaluronic acid) as a major constituent. We speculate that this phenomenon corresponds to an early step of angiogenesis as observed in vivo as a consequence of interleukin release.Because of its unique and strategic position, vascular endothelium participates in a wide range of normal and pathological processes, including inflammation and lymphocyte traffic through specialized postcapillary venules in lymphoid tissue (14, 34). The role played by endothelial cells in these events would be better defined if the factors that modulate their physiological properties were identified.There is increasing evidence that lymphoid cell products may profoundly affect vascular endothelium. Antigen-or mitogen-activated mononuclear leukocytes synthesize and release an array of soluble factors (collectively called lymphokines or interleukins) that have a wide spectrum of regulatory activities on both lymphoid (38, 44) and nonlymphoid cells (33,44,54), and several observations suggest that endothelial cells can also be a target of interleukin activity (2,9,12,13,39,41,49). This prompted us to investigate the effect of interleukins on endothelial ceils in an in vitro system. For this purpose, monolayer cultures of human umbilical vein endothelial (HUVE) ~ cells were incubated with cell-free supernatants from cultures of lectin-activated mononuclear leukocytes and examined by electron microscopy. The results of this study show that interleukins induce cultured HUVE cells to elaborate a highly organized pericellular matrix containing cytochemically recognized glycosaminoglycans (GAG) as a major constituent. MATERIALS AND METHODSCell Culture: Primary cultures of HUVE cells were prepared by an adaptation of previously described methods (17,28). Briefly, the umbilical vein was perfused with 50 ml of Han...

Lymphoid cells differ in the constitution of their surface membranes as demonstrated by study of their surface antigens. Aside from the surface immunoglobulin (sIg), 1 which has been demonstrated on a proportion of lymphoid cells (1, 2), some antigenic determinants of the cell membranes appear to be specifically related to the pathway of differentiation followed by the cells which bear them. These "differentiation antigens" (3) can thus be used to recognize, among lymphocytes, those which are derived from the thymus, or T lymphocytes, and those which are thymus independent, or B lympbocytes. In the mouse, alloantisera raised against the 0 antigen have been widely used for the recognition of T lymphocytes (4). Heteroantisera can also be obtained by immunmation of other species with mouse thymocytes (5) or brain (6); after appropriate absorption on mouse tissues, in vivo (5) or in vitro (6), these antisera recognize antigenic determinants which appear to be present only on T lymphocytes and which have been called "mouse-specific lymphocyte antigens," MSLA (5), brain-associated 0 antigen, BAe (6). It is also possible to obtain, by immunization with mouse lymphoid cells depleted in T cells, heteroantisera which after absorption by mouse thymocytes appear to recognize antigenic determinants present on B but not on T lymphocytes, and called for this reason "mouse-specific bone marrow-derived lymphocyte antigen(s)," MBLA (7).

The role of platelets was investigated in two models of lipopolysaccharide (LPS)-induced toxicity in mice: the systemic reaction, provoked by intravenous LPS injection in D-galactosamine-sensitized recipients, which results in host death, and the local reaction, elicited in the skin by sequential injections of LPS and tumor necrosis factor alpha at 24-h intervals, which results in hemorrhagic necrosis. In both models, the depletion of platelets with a rabbit polyclonal or a mouse monoclonal antiplatelet immunoglobulin G afforded significant protection. In the local reaction, studies of the distribution of "'In-labelled platelets as wvell as optical and electron microscopy showed that platelets are localized in the dermal venules before hemorrhage occurs. Anti-CD11a (LFA-1) and anti-CD54 (ICAM-1) monoclonal antibodies prevented both platelet localization and hemorrhagic necrosis, and these determinants were detected on mouse platelets by immunofluorescence. The antiplatelet monoclonal antibody did not reduce the localization of polymorphonuclear leukocytes in the dermal venules, as shown by histological sections. Thus, in the local reaction, the stimulation with LPS and tumor necrosis factor alpha leads to a binding of platelets to the endothelium of venules by their P2 integrins, which seems necessary for the development of the hemorrhagic necrosis.

The aim of the present study was to quantify in man the distribution and clearance of two aqueous sodium hyaluronate (SH) solutions of 0.125% and 0.250% after the administration of 25 microliters onto the cornea. Isotonic phosphate buffer (PB) was used as a reference instillation. No systemic or local medication was given to the seven 18- to 30-year-old, healthy male volunteers. A detailed evaluation of the anterior segment of the eye, as well as a Schirmer test and a break-up time measurement, yielded results within the normal range. The clearance of 0.125% and 0.250% SH solutions radiolabelled with sodium pertechnetate Tc-99m was measured by gamma scintigraphy and compared with that of a PB solution tagged with the same radiolabel. There was no statistically significant difference between the quantities of 0.125% SH and PB solutions remaining in the precorneal space at 20 min (paired t-test, P = 0.78, n = 7). However, in comparing the 0.250% SH with the PB solution, we observed a statistically significant difference (P = 0.01, n = 7) in the amount remaining in the precorneal space after the same interval. Actually, 53% of the radiolabelled 0.250% SH solution remained on the cornea as compared with 30% for the 0.125% SH solution and 18.3% for the PB solution. These results suggest that an SH solution of 0.250% might have a prolonged residence time on the precorneal surface, and that SH could therefore be used as an additive in various drug-release systems for the eye.