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Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.

Earlier studies on antisera against Fab of pooled human IgG and IgA myeloma proteins disclosed the presence of class-specific Fd antibodies, the demonstration of which required interaction of heavy and light chains. To extend our knowledge about the antigenic structure of the Fd fragment of human immunoglobulins, antisera were prepared in rabbits against gamma chains of pooled IgG and of four IgG1 myeloma proteins, and Fd gamma fragment and a cyanogen bromide-produced CH1 preparation of an IgG1 myeloma protein, and an Fd' alpha fragment of an IgA1 myeloma protein. No antigenic determinants exclusively confined to the CH1 region of intact human IgG could be demonstrated with these antisera. The antigenic structure of CH1 intact immunoglobulins may thus only be defined by light-chain-dependent determinants.

In the present study the activation of purified human T lymphocytes by the calcium ionophore A23187 was analysed in the light of current concepts of receptor-linked inositol lipid metabolism. It was found that A23187 was only slightly mitogenic, with a narrow optimum at 400-500 nM. The proliferation could be blocked by anti-Tac ascites at 10(-3) dilution, suggesting an interleukin 2 (IL-2)-dependent pathway of activation. However, an unexpectedly large proportion of A23187-stimulated cells expressed the IL-2 receptor. Reculturing the cells with exogenous IL-2 after removal of A23187 resulted in strongly enhanced proliferation. Phorbol myristic acetate (PMA) at non-mitogenic concentrations exerted an extremely strong synergistic effect on A23187-induced cell proliferation, which was, again, mediated via an IL-2-dependent pathway. Supernatants of A23187-stimulated T cells did not contain detectable amounts of IL-2. Combination of PMA and A23187 resulted in considerable IL-2 production. It is concluded that A23187 induces the expression of IL-2 receptors without concurrent stimulation of IL-2 production, thus allowing only low levels of proliferation. Addition of exogenous IL-2 or of PMA restores the imbalance between the occurrence of IL-2 and its receptor and results in high rates of proliferation.

Ox erythrocytes, sensitized with IgM-type rabbit antibodies, were found to form rosettes with human blood lymphocytes. Granulocytes, monocytes, and macrophages appeared to lack this property. The rosette formation could be inhibited with human IgM and its Fc fragments. IgM-binding lymphocytes were also present in cell suspensions isolated from human tonsils and thymuses and among blood lymphocytes cultured in vitro. The outcome of combined marker tests and cell separation experiments disclosed that the rosetting lymphocytes belong to the T-lymphocyte population.

With the use of the fluorescent Ca++ indicator Quin-2, we have measured changes in intracellular calcium levels in human B cells in response to anti-Ig antibodies, to Staphylococcus aureus (Staph) or to protein A. Cells of an Epstein-Barr virus-transformed mu lambda-carrying B-cell line, AZU-1, increased free cytosolic calcium after addition of anti-mu or anti-lambda antibodies; F (ab')2 fragments with anti-mu specificity were equally effective. Fab fragments of sheep anti-Ig antibodies only induced a rise in calcium levels after addition of a second anti-sheep Ig antiserum. Cross-linking of non-Ig surface determinants did not influence calcium homeostasis. The calcium channel blockers verapamil (100 microM), nifedipine (20 microM), and LaCl3 (200 microM) inhibited the anti-mu-induced calcium influx. Peripheral blood B cells reacted in essentially the same way in response to anti-mu antibodies. The B cell mitogens protein A and Staph also induced a rise in intracellular calcium. These observations indicate that Ca++ may play a role as a messenger in the activation of human B cells via surface Ig.

When human peripheral blood B cells are cultured for 6 days with the T cell-dependent peptide antigen ovalbumin (OA) in the presence of antigen-presenting cells and helper T cells, plaque-forming cells (PFC) are generated. These OA-induced PFC differ from the conventional high-rate antibody-secreting PFC formed after stimulation of B cells with recall antigens (e.g. tetanus toxoid) in that they secrete antibody at a very low level. Previous studies have shown that OA-induced PFC are B lymphocytes in an early activation state rather than cells that have differentiated into plasmablasts. The apparent arrest in the maturation of OA-induced PFC in an early activation phase can be overcome by simultaneous stimulation with interleukin 2 (IL-2) and gamma interferon (IFN-gamma). The isotype of the OA-specific antibodies secreted, however, are only of the IgM class, demonstrating that an isotype switch does not occur.

Rabbits were hyperimmunized with Micrococcus Pysodeicticus, leading to homogeneous antibody responses. Peripheral blood lymphocytes were taken from the rabbits before and monthly (during 3 months) after the start of the immunization. The cells were stored frozen. Lymphocytes were tested with anti-idiotypic conjugates for the presence of surface idiotypic structures. The nature of the idiotype-positive cells was determined with respect to the presence of IgM or T-cell antigenic determinants on their surface. A sharp rise and fall in the percentage of idiotypic lymphocytes was found, ranging between 1/40,000 and 1/1,000. Initially almost all idiotypic lymphocytes were IgM-positive. In the blood taken 2 months after the start of the immunization 20% of the idiotypic cells belonged to the T-cell population and 10% were negative for both IgM and T-cell antigenic determinants.

In this study the idiotype-positive B cells of one patient with smouldering multiple myeloma (IgG kappa) and of one patient with multiple myeloma (IgG lambda) were analysed phenotypically and functionally. As regards the expression of B cell-associated differentiation antigens and size distribution, the idiotype-positive B cells resembled normal IgG-bearing blood B cells. In functional studies the lymphocytes were cultured in vitro with Staphylococcus aureus, pokeweed mitogen, T-cell factors, or combinations of these. After culture, proliferation and differentiation of the idiotype-positive B cells were measured by autoradiography, an idiotype-specific ELISA, and a spot ELISA. The results show that idiotype-positive B cells of both patients are able to proliferate after stimulation in vitro. In contrast to their normal counterparts, however, almost no increase in the amount of secreted idiotype IgG could be induced. This suggests that the idiotype-positive blood B cells have lost some of their ability to respond to exogenous stimuli.

By means of a panel of monoclonal antibodies it is demonstrated that, in cultures of human peripheral blood mononuclear cells (PBMC) with the T-cell-dependent (TD) antigen ovalbumin (OA), responding B cells are activated from the resting state. The differentiation of the activated B cells to high rate-secreting plasma blasts, however, is arrested in an early activation phase, in which they can be detected as low rate-secreting plaque-forming cells. The arrest does not occur when stimulation with OA occurs in the presence of antigen-nonspecific activation and maturation factors, which are provided in the culture by the anamnestic response to the TD antigen tetanus toxoid.