Effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils.

In vitro studies of neutrophil adhesion generally utilise purified populations and are often performed at 37 degrees C. This study determines the effects of temperature changes and neutrophil separation procedures on the expression of cell adhesion molecules on neutrophils. We found that neutrophil separation procedures involving erythrocyte sedimentation and hypotonic lysis are associated with a significant increase in the expression of both a structural and functional epitope of the beta 2 integrin CD11b, an increase in the expression of sialyl Lewisx (CD15s) and the hyaluronate receptor (CD44) as well as a significant decrease in L-selectin (CD62L) expression. Separated neutrophils are also more resistant than unseparated neutrophils to PMA induced upregulation of a functional epitope of CD11b. Incubating neutrophils at 37 degrees C is associated with increases in the expression of structural and functional epitopes of CD11b. Neutrophil separation is also associated with increases in the expression of both structural and functional epitopes of CD11b which is greater when neutrophil separation is performed at room temperature compared with neutrophil separation at 0-4 degrees C. However, this difference is lost when the latter are incubated at 37 degrees C. Furthermore, neutrophil separation at both 0-4 degrees C and room temperature is associated with a significant increase in CD15s expression. This increase is less when separation is performed at room temperature. These findings suggest that neutrophil separation should be performed at room temperature unless the cells are going to be used at 0-4 degrees C. Researchers using purified neutrophil populations need to be aware of these significant structural and functional changes when extrapolating in vitro results to in vivo situations.

Detection by immunofluorescence of surface molecules present in low copy numbers. High sensitivity staining and calibration of flow cytometer.

Receptors for lymphokines and growth factors are present on cell surfaces often at concentrations of 100-500 copies per cell. Although conventional immunofluorescence cannot detect such low levels, cell membrane antigens present at these concentrations can be detected using an optimally set up flow cytometer together with a three-layer immunofluorescence technique, consisting of monoclonal antibody reacted with selected batches of biotinylated horse anti-mouse immunoglobulin and phycoerythrin-streptavidin. In this study we purified and radiolabelled a number of monoclonal antibodies, determined the specific radioactivity by self-displacement analysis, and used the radiolabelled antibody in experiments where the number of molecules of antibody bound per cell and the fluorescence intensity were measured on the same sample. This permitted us to determine the sensitivity of the fluorescence procedure in molecules per cell, using several different antibody/target cell combinations. The method was consistently capable of detecting fewer than 100 molecules of antibody bound per cell.

A comparison of the sensitivity of immunoperoxidase staining methods with high-sensitivity fluorescence flow cytometry-antibody quantitation on the cell surface.

Surface molecules present in low copy numbers can be detected with high-sensitivity fluorescence flow cytometry. Many cells previously thought not to express certain molecules on their surface can now be shown to have these molecules in very low copy numbers by high-sensitivity fluorescent cytometric methods. Detection of molecules by immunoperoxidase staining methods has not previously been compared with high-sensitivity flow cytometry techniques. Computerized video image analysis (VIA) is a method that allows measurement of area and density of the immunostain chromogen reaction product in a standardized fashion analogous to flow cytometry. In this study, we compared immunoperoxidase reaction products measured by VIA methods with high-sensitivity flow cytometric measurements for cells with 10,000 down to 50 antibody molecules bound to their surfaces. Detection of 100-200 surface molecules was possible with heavy metal-enhanced immunoperoxidase methods, whereas standard immunoperoxidase methods were not as sensitive. The sensitivity of the nickel-enhanced immunoperoxidase staining method was confirmed for detection of an epitope (Tac-IL2 receptor alpha-chain) present in low numbers on the surface of peripheral blood lymphocytes.

Circulating human T and B lymphocytes express the p55 interleukin-2 receptor molecule (TAC, CD25).

Using a highly sensitive immunofluorescence procedure 15-45% of blood lymphocytes from normal human donors can be shown to react with anti-TAC (CD25) monoclonal antibodies. The standard procedure for indirect immunofluorescence does not allow the detection of these CD25-positive cells. Ten healthy adults were tested, and all showed a population of positive cells. Three donors have been tested more than once, and have given consistent results. Three different CD25 antibodies were used, again with consistent results. Double-marker studies showed that the positive cells included B cells, as well as CD4-bearing and CD8-bearing T cells.

Patients with HIV infection have a reduced proportion of lymphocytes expressing the IL2 receptor p55 chain (TAC, CD25).

Using a high-sensitivity immunofluorescence procedure, a proportion of blood lymphocytes can be shown to express the p55 chain of the IL-2 receptor (CD25, TAC) without in vitro stimulation. In this study, we measured the proportion of peripheral blood lymphocytes expressing CD25 in patients with a diagnosis of HIV infection and compared the results with cells from controls. The mean value for HIV-positive samples was 15%, while controls gave a mean value of 31%. The difference between the groups was highly significant (P less than 0.001, Mann-Whitney U test). When expression of CD25 was examined separately in CD4-positive and CD8-positive T cells and in B cells, there was a wider spread of values in the HIV group compared with controls, but no systematic difference. In the patient group studied (110 samples from 53 patients) there was a weak correlation between CD25 and CD4 expression (correlation coefficient r = 0.21, P less than 0.02), but there were patients with low CD25 and high CD4 as well as patients with high CD25 and low CD4, suggesting that CD25 can vary independently of changes in CD4 lymphocytes. Although the majority of very low values of CD25 (less than 10%) were found in patients with stage IV disease, such low values were also common in Stage II disease.

Expression of the Fc receptor for IgG (Fc gamma RII/CDw32) by human circulating T and B lymphocytes.

Tissue-specific isoforms of the human FcR for IgG Fc gamma RII (CDw32) have previously been described by using mAb. These mAb were shown to exhibit different patterns of reactivity with lymphocytes. Among human PBL, Fc gamma RII has been detected on B cells but not T cells when assessed by flow cytometry and microscopy with the use of mAb KB61 and 41H16. Although KB61 and 41H16 were found to react with B cells, the mAb IV.3, CIKM5, and 2E1 did not react with any PBL subset. In this study, we show that KB61 and 41H16 react strongly with the majority (93-96%) of B cells (CD20+), and weakly with a proportion (18-42%) of T cells (CD3+), including 10 to 14% of CD4+ and 27 to 69% of CD8+ cells. In addition, mRNA for Fc gamma RII was detected in purified CD3+CD8high+ lymphocytes by polymerase chain reaction. KB61 and 41H16 also reacted with a majority of CD3-CD16/CD56+ cells, and CD3-CD20- cells. These findings indicate that a subset of T cells and non-T/non-B cells express Fc gamma RII, and are of interest in the light of previous studies which postulate that human Fc gamma R+ cells and Fc gamma RII+ murine T cells suppress the B cell immune response.