Fibrin Induction of Interleukin-8 Expression in Corneal Endothelial Cells In Vitro

Purpose. Classically, acute and chronic inflammations are characterized by fibrin deposition and a dynamic influx of leukocytes. This leukocyte recruitment, and associated activation, is thought to be dependent on the generation of leukocyte chemotactic factors (LCF). Although the functional existence of LCF in ocular tissue has been demonstrated, the identity, source (s), and mechanisms of induction of these LCF are unclear. The authors investigate the hypothesis that in vitro corneal endothelial cells produce LCF in response to fibrin-induced activation. They further hypothesize that in vivo this fibrin-induced expression of LCF contributes to the leukocyte accumulation associated with ocular injury. Methods. Bovine corneal endothelial cells (BCEC) were co-cultured for 3 to 72 hours with physiologic concentrations of highly purified fibrin (0.125 to 2.0 mg/ml) polymerized in situ. At harvest, the conditioned medium was separated from the fibrin matrix by centrifugation and characterized for the presence and nature of polymorphonuclear leukocyte chemotactic activity. This fibrin-induced LCF was compared to known LCF, such as interleukin-8 (IL-8), using standard physical, chemical, and immunologic parameters. Results. Conditioned medium from fibrin-treated BCEC exhibited a dose-and time-dependent induction of LCF activity, as verified by checkerboard analysis. This LCF activity was not immunoprecipitated by a polyclonal antibody to bovine fibrinogen, and it was heat stable (60°C, 90 minutes) and protease labile. Isoelectric focusing and gel filtration analysis revealed a major peak of chemotactic activity at pH 8.5 to 9.0 and a molecular weight of 10 kd, respectively. Radioimmunoassay of conditioned medium from fibrin-treated BCEC for IL-8 demonstrated an 11-fold increase in IL-8 antigen for fibrin-treated BCEC compared to control BCEC. Conclusion. In vitro, fibrin induces BCEC expression of LCF activity that includes IL-8. In vivo, this fibrin induction of LCF from corneal endothelial cells probably serves to control both leukocyte recruitment and activation within the anterior chamber in general and to corneal endothelium in particular. These studies provide a foundation for understanding the nature, sources, and mechanisms of the LCF generation that contributes to endocular inflammation. Invest Ophthalmol Vis Sci. 1994; 35:3980-3990. Inflammatory processes, whether initiated by infectious, immunologic, or environmental factors, represent extremely explosive and potentially destructive biologic responses. Classically, these reactions are characterized by leukocyte influx and fibrin deposi

Impact of Macrophage Deficiency and Depletion on Continuous Glucose Monitoring In Vivo

Although it is assumed that macrophages (MQ) have a major negative impact on continuous glucose monitoring (CGM), surprisingly there is no data in the literature to directly support or refute the role of MQ or related foreign body giant cells in the bio-fouling of glucose sensors in vivo. As such, we developed the hypothesis that MQ are key in controlling glucose sensor performance and CGM in vivo and MQ deficiencies or depletion would enhance CGM. To test this hypothesis we determined the presence/distribution of MQ at the sensor tissue interface over a 28-day time period using F4/80 antibody and immunohistochemical analysis. We also evaluated the impact of spontaneous MQ deficiency (op/op mice) and induced-transgenic MQ depletions (Diphtheria Toxin Receptor (DTR) mice) on sensor function and CGM utilizing our murine CGM system. The results of these studies demonstrated: 1) a time dependent increase in MQ accumulation (F4/80 positive cells) at the sensor tissue interface; and 2) MQ deficient mice and MQ depleted C57BL/6 mice demonstrated improved sensor performance (MARD) when compared to normal mice (C57BL/6). These studies directly demonstrate the importance of MQ in sensor function and CGM in viv

Effect of Lipopolysaccharide on C3 and C5 Production by Human Lung Cells

Although studies to date have demonstrated the ability of the monocyte/macrophage to produce C components in vitro, very few studies on C production by nonhepatic tissue cells have been reported. Recently, using 35S-methionine incorporation and immunoprecipitation techniques our laboratory has demonstrated the ability of tissue cells, i.e., the human lung type II pneumocyte (A549) and human lung fibroblast (WI-38), to synthesize and secrete a variety of early and terminal complement components, as well as several regulatory proteins in vitro, i.e., C1r, C1s, C4, C3, C5, C6, C7, C8, C9, factor B, factor H, factor I, and C1s inactivator. In our studies, we extended these observations by demonstrating the capability of LPS to modulate C3 production by A549 pneumocytes. Specifically, using a sensitive ELISA we demonstrated that A549 pneumocytes exposed to LPS induced an 80 to 180% increase in C3 levels when compared to untreated A549 cells. Interestingly, LPS had no effect on C5 production or total protein synthesis by A549 pneumocytes. In the case of the WI-38 fibroblast, LPS had no effect on 1) C3 production, 2) C5 production, or 3) total protein synthesis in vitro. These studies demonstrate that agents such as LPS have the potential to selectively regulate C production (i.e., C3) in individual lung cells in vitro, and suggests that in vivo LPS may alter the local tissue reservoir of C components during infection and lung injury, thus impacting on pulmonary inflammation and host defense.

Silica Induced Suppression of the Production of Third and Fifth Components of the Complement System by Human Lung Cells In Vitro

Although investigations to date have demonstrated the ability of the monocyte/macrophage to synthesize complement components, only a limited number of studies on complement synthesis by nonhepatic tissue cells have been reported. To begin to fill this gap in our knowledge we have recently evaluated the ability of lung tissue cells to synthesize and secrete various complement components in vitro. Using 35S-methionine incorporation and immunoprecipitation techniques we have previously demonstrated the ability human lung type II pneumocytes (A549) and human lung fibroblasts (WI-38), to synthesize and secrete a variety of both early and terminal complement components, as well as several regulatory proteins including C1r, C1s, C4, C3, C5, C6, C7, C8, C9, Factor B, Factor H, Factor I and C1s inactivator. Our present studies demonstrate the capability of silica to regulate complement component production by A549 cells, but not complement component production by WI-38 cells. Specifically, using sensitive ELISAs we demonstrated that a non-toxic dose of silica had the capability to suppress the production of both C3 and C5 by A549 pneumocytes by 40-50 percent, but had no effect on C3 or C5 synthesis by WI-38 fibroblasts. Additionally, using 35S-methionine incorporation and TCA precipitation techniques, we demonstrated that suppression of C3 and C5 production by silica treated A549 pneumocytes was not a result of suppression of total protein synthesis. These studies demonstrate that silica, which has been implicated in pulmonary diseases, has the capability to regulate local complement production by lung tissue cells in vitro. In vivo, this suppression of complement production by the type II pneumocytes could alter the local tissue reservoir of complement components during infection and pulmonary injury, thus resulting in depressed pulmonary host defense.