T cell--vascular smooth muscle cell interactions: antigen-specific activation and cell cycle blockade of T helper clones by cloned vascular smooth muscle cells.

Histological observations have demonstrated the presence of T lymphocytes in atherosclerotic plaques, often in close association with vascular smooth muscle cells (VSMC). We have examined the interaction occurring between cloned murine VSMC and histocompatibility-matched, antigen-specific Th1 and Th2 cell lines. Incubation of either Th1 or Th2 cells with antigen-pulsed VSMC resulted in the formation of T cell-VSMC conjugates accompanied by morphological changes in both cell types. This interaction resulted in an antigen-dependent activation of IL-2 receptor expression by the Th cells, demonstrating the ability of cloned VSMC to process and present antigen through the exogenous pathway. However, although the T cells were activated to express IL-2 receptors by antigen-pulsed VSMC, they were unable to progress through cell cycle. The secretion of an inhibitory mediator by VSMC was suggested by the observations that (1) fixation of the VSMC's eliminated the inhibitory signal and (2) the supernatants of IFN gamma-primed VSMC displayed similar inhibitory activity. The inhibitory effect could not be abrogated with indomethacin or an inhibitor of the generation of reactive nitrogen intermediates, indicating that prostaglandin synthesis and/or nitric oxide production are not solely responsible for the inhibition of proliferation. Flow cytometric cell cycle analysis revealed that VSMC delivered signals resulting in a late G1 blockade of T cell cycle progression. Mitogen responses of purified primary T cells are also dramatically inhibited by IFN gamma-treated VSMC, despite significant IL-2 production. Our data depict a complex and intimate T cell-VSMC interaction and suggest that mutual activation events may occur.

Cloned endothelium derived from autoimmune vascular disease retain structural and functional characteristics of normal endothelial cells.

MRL/1pr mice demonstrate anatomic specificity in their development of vasculitis including the small- and medium-sized muscular arteries of the mesentery. To define the functional role of endothelium in vasculitis, we have cloned endothelial cells derived from inflamed small- and medium-sized arteries. Primary cells were derived by enzymatic dispersement and endothelial cells were selected by utilizing a combination of specific culture conditions. Cloned endothelium were developed utilizing limiting dilution cultures supplemented by endothelial cell growth factor. The cloned endothelial cells express many structural features of mature endothelial cells including Factor VIII-RA, non-muscle-specific actin, and Weibel-Palade bodies. Functionally, the clones express functional receptors for the scavenger pathway for LDL metabolism. The cells do not express Class I MHC antigens; however, IFN-beta and IFN-gamma stimulate Class I MHC expression after 24 h, which induces lysis of virus-infected cloned endothelium by Class I-restricted virus-primed T cells. In direct contrast to site-identical vascular smooth muscle cells (VSMCs), endothelial cells do not spontaneously express Class II MHC antigens, nor do they secrete biologically relevant levels of IL-1 unless triggered by lipopolysaccharide. The availability of site-specific cloned endothelium along with cloned VSMCs from autoimmune mice should resolve major experimental controversies involving the pathophysiology of inflammatory vascular disease.

Autoimmune vasculitis in MRL/Mp-lpr/lpr mice: orthochromatic basophils participate in the development of delayed-type hypersensitivity angiitis.

The pathogenesis of autoimmune vasculitis is poorly understood. Understanding the immunologic mechanisms governing this disease requires precise identification of the cells which comprise the lesion. In this report, we have evaluated tissue sections from MRL/lpr mice from 16 to 45 weeks of age, representing all stages of clinical vasculitis. We demonstrate that basophil myelocytes participate in the evolution of the delayed-type hypersensitivity (DTH) response which initiates and perpetuates autoimmune vasculitis in these mice. These findings raise questions regarding the immunologic mechanisms by which basophils develop in this lesion and the interaction of basophils. VSMCs and lymphocytes in vasculitic angiodestruction.

Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis.

Interleukin-1 (IL-1) has been implicated as a regulatory protein in the development and clinical sequelae of atherosclerosis. To determine which cells in the atherosclerotic plaque synthesize IL-1 in situ, the authors evaluated histologic sections of iliac arteries from cynomolgus monkeys using probes for IL-1 alpha and beta. A polyclonal antibody to IL-1 alpha and beta was used to determine if proteins were concomitantly produced. The predominant cells expressing IL-1 alpha and beta mRNA were foam cells in the intima. Adherent leukocytes and vascular smooth muscle cells (VSMCs) expressed mRNA for IL-1 alpha. Microvascular endothelium expressed mRNA for both IL-1 alpha and beta. IL-1 proteins were located frequently in cells expressing IL-1 mRNA. These results indicate that endothelium and VSMCs, in conjunction with macrophages, serve as localized sources of IL-1 protein synthesis. These findings suggest that vascular cells may contribute directly to the pathogenesis of atherosclerotic vascular disease by actively secreting potent biologic mediators that modify vascular and immune cell function.

Vasculitis in MRL/1 pr mice: model of cell-mediated autoimmunity.

The destruction of vascular smooth muscle cells (VSMC) in autoimmune arteritis is a poorly understood phenomenon. To approach this problem, VSMC cultures were established. The interaction of these cells (from normal or autoimmune mice) with lymphocytes was then evaluated. Specifically, splenocytes from MRL/1pr or C3H mice were co-cultivated with MRL/1pr or C3H VSMCs. Massive mononuclear inflammatory cell clusters enveloped MRL/1pr VSMCs which culminated in the detachment of MRL/1pr VSMCs from the culture plate. In contrast, the interaction of splenocytes from normal or autoimmune mice did not destroy normal VSMCs. Further investigation indicated that MRL/1pr VSMCs spontaneously expressed both Ia-k and Ia-d, as assessed by fluorescence microscopy and flow cytometry, and released interleukin-1-like factors--characteristics of accessory cells to T-lymphocyte function. Evaluation of VSMCs accessory function in antigen presentation suggests that these cells may present antigen under specific experimental conditions. As a result of these studies, a novel mechanism of autoimmune vasculitis is proposed. Our hypothesis is that defective biological function of VSMCs from autoimmune mice stimulates a mononuclear inflammatory cell response which culminates in VSMC autodestruction.

Venular endothelium in vitro: isolation and characterization.

The structural and functional properties of the endothelium vary in relation to anatomic site and position along the vascular tree. Cultures of endothelial cells have been obtained so far from large arteries, large veins and capillaries, but not from venules. We now report techniques for culturing not only rat arterial and venous endothelium, but also a special method for obtaining and culturing venular endothelium. The technique is based on the principle of "vascular labeling," whereby an insoluble pigment can be permanently deposited in the wall of the venules, making them easily visible by light microscopy. The venules of a rat cremaster muscle are labeled with a local injection of histamine followed by Monastral blue B intravenously (i.v.); 24 hours later selected venules are isolated by microdissection and either enzymatically dispersed or placed directly into tissue culture wells. The wells are coated with fibronectin and laminin and supplemented with DMEM, 20% fetal calf serum, and endothelial cell growth factor. Polygonal and spindly endothelial cells begin as clusters, grow in sheets, and sometimes form tubes. The cells stain variably for Factor VIII-related antigen, Ulex Europeus I lectin, and non-muscle specific actin. They synthesize angiotensin-converting enzyme, but do not metabolize acetylated LDL. Ultrastructurally, they display pinocytic vesicles, microtendons, and tight junctions, but not Weibel-Palade bodies. We believe that this method will be important for studying the pathophysiology of venules, which are the preferential target of inflammatory mediators and the typical site of inflammatory cell diapedesis.

Systemic mononuclear-cell vasculitis in MRL/Mp-lpr/lpr mice. A histologic and immunocytochemical analysis.

The cellular mechanisms governing the expression of mononuclear cell vasculitis are poorly understood. For determination of the precise sequence of events in the development of vasculitis in autoimmune MRL/lpr mice, histologic sections from 4-20-week-old mice were evaluated with a panel of cytochemical and immunohistochemical stains. The results show that vascular disease in MRL/lpr mice develops as follows: Thy 1+, Ly 1+, L3T4- T cells assemble around predominantly small-to-medium muscular arteries at approximately 8 weeks of age. At 12 weeks of age, an adventitial inflammatory focus forms, composed of large "reactive" mononuclear inflammatory cells adjacent to hypertrophied vascular smooth muscle cells (VSMCs). Blastic Thy 1+, Ly 1+, L3T4- T cells subsequently infiltrate the tunica media, and selective VSMC karyolysis results. Occasional cytotoxic/suppressor T cells, macrophages, and possibly NK cells are noted primarily distal to the infiltration site. The outer zone of the inflammatory infiltrate is composed of mature B cells and occasional B-cell precursors. These findings suggest that cellular constituents of the immune response mediate mononuclear cell vasculitis in MRL/lpr mice.

Growth and analysis of vascular smooth muscle on microspheres.

Protocols for culturing vascular smooth muscle cells (VSMCs) on polystyrene microspheres, assessing VSMC contractility while adhered to the microspheres, and analyzing VSMCs cultured on microspheres by flow cytometry are described. These procedures preserve optimal VSMC viability and function, while providing a method for assessing these cells within the parameters required for analysis by flow cytometry.

The role of vascular smooth muscle cells in experimental autoimmune vasculitis. I. The initiation of delayed type hypersensitivity angiitis.

The destruction of vascular smooth muscle cells (VSMCs) in autoimmune arteritis is a poorly understood phenomenon. For evaluation of the cellular interactions that may contribute to vasculitis, the immunobiology of VSMCs and lymphocytes was explored in vitro. Primary VSMC cultures were established, and the interaction of these cells (from normal or autoimmune mice) with lymphocytes was then assessed. Specifically, splenocytes from MRL/lpr or C3H mice were cocultivated with MRL/lpr or C3H VSMCs. Massive mononuclear cell clusters from normal and autoimmune mice enveloped MRL/lpr VSMCs, which culminated in the detachment of MRL/lpr VSMCs from the culture plate. In contrast, the interaction of SPs from either normal or autoimmune mice did not encompass or destroy normal VSMCs. Further investigation indicated that MRL/lpr, but not C3H, VSMCs spontaneously expressed Ia and released Il-1 like factor(s), which may be at least two mechanisms by which MRL/lpr VSMCs stimulate the in vitro mononuclear cell influx. As a result of these studies, a novel mechanism for the induction of mononuclear cell autoimmune vasculitis is proposed. VSMCs derived from autoimmune mice may stimulate a mononuclear inflammatory cell phlogistic response which culminates in VSMC autodestruction.

Improved healing of experimental long bone fractures in rabbits by delayed internal fixation.

Experiments in rabbits were conducted to test the clinical propositions that a) the risk of nonunion in certain long bone fractures can be sharply reduced by open reduction and internal fixation, and b) that there is an optimum critical period following trauma in which this procedure should be carried out to facilitate healing. Bilateral standard open fractures of rabbit radii were internally fixed by intramedullary K-wires immediately or at 5, 10, and 17 days after trauma. These bones were subjected to stress and histometric analyses at 4-56 days after the initial fractures. The observations suggested that the pace of fracture healing could be optimized by time-delay surgery. The most exuberant and strongest calluses developed when the bones were fixed 10 days after fracture, and the rate of healing was improved. Delay times of 5 or 17 days did not provide better healing than that achieved by an immediate operation. Callus quality, in terms of the proportion of fiber bone, lamellar bone, and cartilage, was similar in all groups. The tensile strengths of the bones during callus formation were highly correlated with the ratio of callus/cortical bone areas (p less than 0.01).