Thymic epithelium in vitro. IV. Regulation of growth and mediator production by epidermal growth factor.

The regulation of thymic epithelial cell function has been examined using pure cultures of morphologically distinct thymic epithelial cells and the ubiquitous hormone epidermal growth factor (EGF). Small thymic epithelial cells, TECS, had receptors for EGF with high affinity, Kd = 1.2 X 10(-9) M, and exhibited increased DNA synthesis and increased RNA synthesis upon stimulation with EGF. In addition, incubation of TECS monolayers with EGF resulted in enhanced production of prostaglandin E2. In contrast, large thymic epithelial cells, TECL, did not express receptors for EGF and demonstrated no biological response to the hormone. These results suggest the possibility that intrathymic regulation of lymphoid cells may occur via the action of "nonimmunologic" mediators on thymic epithelial cells. They further suggest the more general possibility that immunologic and nonimmunologic hormonal systems may be linked via intersecting cellular pathways.

Substrate requirements for the isolation and purification of thymic epithelial cells.

Cultures of thymic epithelial cells were initiated and maintained in vitro using surfaces coated with different substrates. The substrates we compared were plastic commercially treated for tissue culture, plastic coated with gelatin, and plastic coated with Cell-TakTM, a new commercially available cell and tissue adhesive. Cell-TakTM-treated plates provided the best environment for isolation and purification of this cell type as judged by lack of fibroblast contamination, growth rate, and cell morphology.

Adherence of tumor cells to endothelial monolayers: inhibition by lymphokines.

Tumor cells can adhere to endothelial cell monolayers in vitro. The kinetics of this reaction are rapid; 50% of maximal binding occurs by 30 min of incubation. In the case of the P815 mastocytoma, the maximal percentage of binding is approximately 70%, suggesting that there are both binding and nonbinding tumor cell populations. Binding is independent of tumor cell dose over a 200-fold range of cell concentrations. Lymphokine-containing preparations were found to markedly suppress the binding of either P815 mastocytoma or Ehrlich ascites cells to endothelium. This effect appeared to be due to both diminished attachment and enhanced dissociation. The activity is found in the same molecular weight range as tumor migration inhibition factor (TMIF), and is not found in preparations lacking TMIF activity. Thus, the factor may prove to be TMIF itself or a lymphokine related to it. Of equal interest is the possibility that it represents a previously undescribed factor.

In vitro growth and maintenance of two morphologically distinct populations of thymic epithelial cells.

Cultures of thymic epithelial cells were generated and maintained in valine-free minimum essential medium (MEM) supplemented with 690 mg/liter of D-valine. These cultures have been maintained for 1 year through multiple passages by trypsinization of 60-70% confluent monolayers. Large and small epithelial cells were present in early cultures. They were separated into two stable subpopulations based on (1) their differential growth rates and (2) their differential adherence to the culture substratum. These morphologically distinct cell populations, TECS and TECL, were 100% keratin positive and contained cells with desmosomes and tonofilaments, all characteristics of epithelial cells. Esterase analysis of both cell populations revealed a 1 and 9% esterase-positive cell population in cultures of keratin-positive small (TECS) and large (TECL) cells, respectively. The percentages of esterase-positive cells corresponded to the 2 and 10% populations of TECS and TECL, respectively, that contained both desmosomes and phagolysosomes. These results establish conditions for the long-term propagation of pure thymic epithelial cells. Such cultures can be used to study the functional interactions between epithelial cells and lymphoid cells. Morphologic and histochemical analyses have identified subsets of these cells which may prove to have differential effects on thymocyte proliferative and developmental processes.

Influence of anti-endothelial cell antibodies on the interactions of leukocytes with vascular endothelial cells in vitro.

The role of mononuclear cells in antibody-mediated endothelial cell damage and vascular diseases is not well understood when compared to our understanding of the role of neutrophil-mediated endothelial injury in vascular diseases. Thus we initiated these in-vitro studies to investigate the interactions of leukocytes (neutrophils and mononuclear cells) with vascular endothelial cells in the presence and absence of antiendothelial cell antibodies (anti-red blood cell antiserum-anti-RBC). Cultured endothelial cells were isolated from bovine pulmonary artery. The effects of unfractionated and fractionated human leukocytes (neutrophils, mononuclear cells, or lymphocytes) on endothelial cell viability was examined both quantitatively (51Cr release) and qualitatively (electron microscopy). Results from these studies indicated that the interactions of mononuclear cells and lymphocytes with antibody-coated endothelial cells resulted in a significant endothelial cell lysis within 1 to 6 hours of reactions. Neutrophils, on the other hand, failed to induce significant endothelial cell damage compared to mononuclear cells and lymphocytes when tested under similar conditions. In the absence of anti-RBC antibodies, all cell types (neutrophils, mononuclear cells, or lymphocytes) did not produce detectable endothelial damage. Additionally, the effectiveness of mononuclear cells to injure antibody-labeled endothelial cells was confirmed by ultrastructural examination. Similar studies, utilizing leukocytes obtained from patients with atherosclerosis disease, were also undertaken. In these studies we found that, again, only mononuclear cells and lymphocytes wer capable of inducing damage to endothelial cells precoated with antibodies. In summary, our results demonstrate the ability of mononuclear cells and lymphocytes to induce significant damage to antibody-coated endothelial cells. This finding suggests a major role of mononuclear leukocytes in vascular endothelial destruction in diseases that are characterized by the presence of circulating autoantibodies in serum and the adhesion of mononuclear cells to the vascular wall. Such diseases include vasculitis, allograft rejection, serum sickness, and perhaps atherosclerosis particularly in patients with autoimmune diseases.

Fibrin-mediated vascular injury. Identification of fibrin peptides that mediate endothelial cell retraction.

The deposition of fibrin, a ubiquitous component of acute and chronic inflammatory reactions, has been implicated by a number of recent studies as playing an active role in inflammation. In particular, fibrin deposition has been implicated in the development of tissue edema. As the "gateway" through which intravascular-to-extravascular movement of fluid, nutrients, and cells must pass, the vascular endothelial cells play a crucial regulatory role in this process. In support of this concept, recent studies in this laboratory have demonstrated that endothelial cells retract not only in the presence of fibrin but also in the presence of low molecular weight cleavage products of fibrinogen. It was further shown that this reaction was 1) specific for both vascular and corneal endothelial cells, 2) nontoxic, and 3) completely reversible. The present work examined the physiochemical nature of these endothelial-cell reactive factors. It was demonstrated by the use of enzymatically derived and synthetic fibrinogen peptides, that the active soluble fibrinogen-derived factor was associated with the amino-terminal end of the B chain of fibrinogen. The active factor has been tentatively identified as the B beta peptides, which is a primary plasmin cleavage product of fibrinogen and contains the thrombin-generated fibrinopeptide B. It is thus suggested that soluble, endothelial-cell-reactive peptides are released during both fibrinogenesis and fibrinolysis and, as such, modulate endothelial cell functions in vivo.

Effects of D-valine on pulmonary artery endothelial cell morphology and function in cell culture.

The effects of D-valine on the cell culture of bovine pulmonary artery endothelial cells were studied using D-valine-modified Minimal Essential Medium (MEM). D-Valine-treated cultures (46-920 mg/l) were compared with replicate cells grown in L-valine (46 mg/l)-MEM. All media were supplemented with 15% fetal bovine serum (FBS). Endothelial cells were grown for 14 passages with split ratios varying from 1:3 to 1:6. Unlike cells grown in L-valine MEM, cells grown in D-valine MEM did not become contaminated by the growth fibroblasts in primary cultures. D-Valine-treated cells were found to grow in cobblestone array, exhibit contact inhibition and strongly express factor-VIII antigen (F-VIII). D-Valine-grown cells produced PGI2 in greater proportion to PGE2, both constitutively and when stimulated by bradykinin, on comparison with cells grown in L-valine. In addition, cells grown in L-valine, although able to express factor VIII, were not comparable to D-valine cells with respect to other parameters assayed (morphology and growth as a monolayer).

Fibrin-mediated vascular injury: demonstration of vascular endothelial cell retraction in response to soluble fibrin-associated factors.

Until recently, fibrin deposition has been generally viewed as a ubiquitous, but passive, characteristic of both acute and chronic inflammatory processes. An increasing number of studies, however, suggest that fibrin deposition and fibrin degradation products may play an active role in these reactions, especially in the development of tissue edema. As the intravascular-to-extravascular "gateway" for movement of fluids, solutes, and cells must pass, the vascular endothelial cells may play a crucial regulatory role in this process. Recent evidence suggests that the interaction of whole fibrin with endothelial cells may lead to endothelial cell injury. This work examines the role of fibrin and fibrinogen-associated factors (FAF) in the injury of cultured vascular endothelial cells (VEC). It was demonstrated that endothelial cell injury, evidenced by retraction of the cells, was induced not only by whole fibrin, but also by soluble thrombin-cleaved products of fibrinogen. This reaction was shown to be specific for endothelial cells, nontoxic, and completely reversible. The general biological importance of this reaction was further underscored in that active factors could be generated from fibrinogen obtained from a number of species. Reproducible, quantitative assays of cultured endothelial cell retraction were developed to aid in the characterization of the endothelial cell-specific FAFs.

Migration inhibition of endothelial cells by lymphokine-containing supernatants.

Many of the reactions of cellular immunity are mediated by soluble lymphocyte-derived factors (lymphokines). One important category of lymphokine action involves effects on cell motility. These effects have been described mainly with respect to inflammatory cells. In this report, we describe the ability of a lymphocyte product to inhibit the migration of endothelial cells in a system in vitro. The responsible factor is distinct from a previously described mediator that inhibits the migration of tumor cells. The ability of lymphocytes to influence the migration properties of endothelial cells is consistent with data of others showing a relation between the immune system and processes involving neovascularization.