A fluorescent cellular adhesion assay using insect cell produced human VCAM1.

Activated endothelium and some dendritic cells express the adhesion molecule VCAM1, a member of the immunoglobulin gene superfamily. Mononuclear leukocytes display the integrin VLA4 that functions as a counterreceptor for VCAM1. The interaction of VCAM1 with VLA4 mediates cell to cell adhesion events believed to be important regulators of inflammation, cancer cell metastasis, and atherosclerosis. This report describes the development of a fluorescent adhesion assay that specifically measures T cell adhesion to recombinant human VCAM1 (rVCAM1) expressed in a baculovirus expression vector system (BEVS). We describe a simple and rapid protocol to partially purify non-denatured rVCAM1 from insect cell membrane preparations (VCAM1 infected Sf9 cells). Jurkat cells, a T cell line expressing VLA4, specifically adhered to the rVCAM1 membrane preparations coated onto 96-well plates. Jurkat cells did not adhere to control membrane preparations that lacked rVCAM1 protein. Both unstimulated and IL-2 stimulated Jurkat cells displayed functional VLA4 capable of binding to immobilized rVCAM1. Monoclonal antibodies recognizing either VCAM1 (E1/6, BBA6) or VLA4 (HP2/1) blocked specific VCAM1/VLA4 adhesion, whereas a monoclonal antibody to the alpha chain of LFA1 did not block adhesion. The methods described here could be applied to develop similar functional assays for other cell surface receptors/counterreceptors expressed in a BEVS.

Evidence that increases in lymphocyte tyrosine phosphorylation precede cardiac allograft rejection. Effects of cyclosporine and potential use in clinical management.

Tyrosine phosphorylation is an early, critical event in lymphocyte signal transduction. We measured tyrosine phosphorylation in a porcine experimental transplant model to evaluate its utility in monitoring the allograft immune response. Using flow cytometry, we demonstrate a biphasic increase in phosphotyrosine (ptyr) levels in peripheral blood mononuclear cells (PBMC), and that increases are detectable as early as 1 day posttransplantation in untreated transplanted animals (n = 4). This biphasic response is likely result from the sequestration of ptyr+ cells from the periphery into the graft as graft-infiltrating lymphocytic cells show increased ptyr levels. This suggests possible lymphocyte trafficking between the peripheral compartment and the allograft. A 5-day course of treatment with cyclosporine (CsA) at 20 mg/kg/day (n = 4), but not at 10 mg/kg/day (n = 4), prevents graft rejection in this allograft model. Strikingly, treatment with 20 mg/kg/day CsA, but not with 10 mg/kg/day, suppressed increases in ptyr levels in both PBMC and graft-infiltrating cells. Increases in ptyr levels in PBMC are detectable 2-5 days before histologic and electrocardiographic signs of graft rejection, suggesting a potential diagnostic utility for measuring tyrosine phosphorylation in monitoring and managing transplant rejection.

Identification of a subpopulation of reactive large granular mononuclear cells in allogeneic heart transplantation.

This study is designed to test the hypothesis that specific morphologic attributes of peripheral blood mononuclear cells, measurable by flow cytometry, are correlated with the timing and the intensity of allograft injury during the development of heart rejection. A pig model of major histocompatibility complex-mismatched heterotopic heart transplantation with (n = 5) and without (n = 5) cyclosporine administration was monitored serially be telemetered electrocardiography and endomyocardial biopsies. Flow cytometric analysis of peripheral blood mononuclear cells revealed the emergence of a discrete subpopulation of peripheral blood mononuclear cells (7.8% +/- 1.0% and 8.5% +/- 0.9% before transplantation to 16.5% +/- 1.3% and 19.4% +/- 3.0% after transplantation in the untreated and the cyclosporine-treated groups, respectively, p < 0.05), exhibiting characteristic changes in forward and 90-degree light scatter, indicative of increased cell size and granularity, and possibly representing monocytes or large granular lymphocytes. Lymphocyte cell surface-marker studies indicated that 62% of these cells are DH59B+ (monocyte/granulocyte). Because intracellular free calcium is an important second messenger in lymphocyte activation we measured intracellular free calcium by flow cytometry using fluo-3. This subpopulation of cells was found to have similar intracellular free calcium when compared to normal-sized lymphocytes (104 +/- 7 nmol/L versus 101 +/- 5 nmol/L, respectively). We conclude that this lymphocyte subset detected by flow cytometry represents specifically reactive cells that are associated with incipient allograft rejection.