Synovial fluid cells in juvenile arthritis: evidence of selective T cell migration to inflamed tissue.

The perpetuation of chronic synovitis in juvenile arthritis (JA) is a complex interaction of local and systemic regulatory mechanism. We examined the cell surface phenotype of synovial fluid cells and peripheral blood lymphocytes from 15 patients with JA to better understand the mechanism of local inflammation. Synovial fluid and peripheral blood mononuclear cells were analysed for cell surface expression of CD2, CD3, CD4, CD8, CD19, CD25, CD29, CD45R and Ia using flow cytometry. We found a very low percentage of B cells with a concomitant increase of T cells in synovial fluid as compared with peripheral blood. A large percentage of the synovial fluid T cells were HLA-DR+, or activated T cells, and there was a relative decrease in CD4+ cells in synovial fluid as compared with peripheral blood. There was only a minimal increase in CD25+ synovial fluid cells. The synovial fluid CD4+ cells were mainly of the CD2high, CD29+, CD45RO phenotype. This CD4 phenotype found on synovial fluid cells from patients with JA and in particular the CD29 cell surface marker, which recognizes a common beta-chain of adhesion molecules, is associated with binding to extracellular matrix proteins and is also associated with 'primed' T cells. Our results demonstrated the presence of T cells which either selectively migrate to synovium and synovial fluid or are activated in situ in the joint.

Interleukin 6 induces myeloid differentiation of a human biphenotypic leukemic cell line.

The human leukemic cell line B1, is characterized by a specific 4;11 chromosomal translocation, immature myeloid/pre-B biphenotypic features, expression of multiple cytokine receptors and IL-1-dependent autocrine growth regulation [Cohen et al. (1991) Blood 78, 94]. Exposure of B1 cells to low concentrations of IL-6 abolished the leukemic cells ability to form colonies in semi-solid medium and slowed down their proliferation rate in suspension. Associated with these changes in growth characteristics, the B1 cells differentiated along the myeloid lineage as judged by the induction of the myeloid-specific surface antigens CD33, CD13 and CD11b, as well as histochemical and morphological changes characteristic of myeloid cells. The induction of differentiation was specific to IL-6 since none of the other cytokines which inhibited B1 cell growth (IL-7, gamma IFN and TNF alpha) were able to induce myeloid or lymphoid differentiation in these cells. The IL-6-induced differentiation was completed over a two week period and was essentially irreversible. Together with the phenotypic changes, IL-6 induced the expression of the protein tyrosine phosphatase (CD45) which may be associated with altered growth observed in IL-6-treated cells. Induction of terminal differentiation of leukemic cells by recombinant bioregulators has therapeutic implications and merits further study.

S100 protein expression in human melanoma cells: comparison of levels of expression among different cell lines and individual cells in different phases of the cell cycle.

The synthesis of S100 protein in cultured human melanoma cells was examined using metabolic labeling with [35S]methionine, immunoprecipitation with anti-S100 protein antiserum, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Six of seven cell lines derived from melanomas synthesized relatively large amounts of S100 protein, whereas three cell lines derived from normal melanocytes synthesized lesser amounts. Synthesis of S100 protein was not detected in 10 human cell lines of nonneuroectodermal origin. Analysis of poly(A+) RNA from one melanoma cell line by Northern blot hybridization with a probe specific for the beta subunit of rat S100 protein revealed a single mRNA species of 1.0 kb coding for the human protein. Flow cytometric analysis of individual cells of two melanoma cell lines and the rat glioma cell line C6 indicated that G0/G1 cells were heterogeneous with respect to S100 protein expression, while almost all the cells in S + G2 + M expressed S100 protein. These results suggest that expression of S100 protein in G0/G1 could be a prerequisite for progression of the cells through the cell cycle.

Quantitative Phenotyping of Childhood Leukemia Identifies Variable and Invariable Cell Surface Antigens.

Cells obtained from 75 cases of childhood leukemia were subjected to flow cytometry analysis to estimate the density of several cell surface antigens and derive a quantitative immunological phenotype. Sixty-five cases of acute lymphoblastic leukemia (ALL) including 10 T-ALL, 6 non-T ALL designated groups I and II (HLA-DRCALLA), 48 non-T ALL termed groups III and IV (HLA-DRCALLA) and one B-ALL were studied; 10 cases of acute myeloblastic leukemia (AML) were also analysed. The estimation of the relative fluorescence index (RFI) on leukemic blasts led to the derivation of mean values for each marker in the leukemia subgroups. We have quantitated the levels of the antigens generally used in the classification of these leukemias (CALLA, CD5, CD20, CD13, HLA-DR and CD19) and of other cell surface antigens associated with leukemic cells. For example, CALLA (CD10) level was high (mean RFI value of 26.4) on the leukemic cells of non-T ALL groups III and IV. The CD5 antigen was present on T-ALL, as expected, with an RFI value of 4.5; however, low levels were observed on the more immature non-T ALL of groups I and II (RFI = 2.3 on only 27% of blast cells). The quantitative analysis of the cell surface antigens associated with non-T ALL has revealed molecules such as CALLA, HLA-DR, CD9 and CD44 present at high and variable levels and others such as CD19, CD38, 44G4, 44D7, 44H9 and 44H6 generally of lower intensity, less variable from one patient to another, and with similar mean levels of expression in the different subgroups. These invariable antigens are not altered by the lineage or stage of differentiation of the leukemic cells. The variable antigens could be correlated with the functional and/or differentiation status of the cells and could also be modified by the alterations of regulatory processes associated with malignancy. The quantitation of multiple leukemia-associated antigens, whose structure and function are becoming rapidly established, should help in elucidating the function of these molecules in leukemogenesis and/or disease progression.

Interferon and phorbol esters down-regulate sIgM expression by independent pathways.

We studied the effects of recombinant interferon, 12-0-tetradecanoylphorbol-13-acetate (TPA), and phorbol 12, 13 dibutyrate (PDB) on surface immunoglobulin expression by Daudi cells. Incubation of cells with recombinant alpha 2 interferon (IFN-alpha 2) caused a 2.5-fold (60%) decrease in sIgM expression as measured by relative fluorescence index (RFI) using a flow cytometer. This decrease in sIgM expression was independent of inhibitory effects on proliferation and cell cycle progression. TPA or PDB also caused a threefold (67%) decrease in sIgM expression, while enhancing proliferation and cell cycle progression. Coincubation of cells with IFN-alpha 2 and TPA decreased sIgM expression by more than fourfold (greater than 75%), which was greater than the decrease induced by the optimal concentration of either agent alone. Molecular studies demonstrated that the treatment of cells with IFN-alpha 2 or TPA decreased the steady-state levels of mRNA for the heavy chain of IgM (c mu), suggesting that down-regulation of sIgM occurred at a pretranslational level. Activation of the cell membrane sodium/proton antiport did not play an integral role in the IFN-alpha 2 or phorbol-ester-induced pathway of sIgM down-regulation. Whereas IFN-alpha 2 induced an increase in the activity of 2',5'-oligoadenylate (2-5A) synthetase, the addition of TPA to IFN-alpha 2 caused a significant decrease in the activity of this enzyme. Although IFN-alpha 2 and TPA exhibited additive effects on sIgM expression, they had opposing effects on cell proliferation, cell cycle progression, and induction of 2-5A synthetase activity, suggesting that these agents down-regulate sIgM expression through independent pathways.

Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells.

We have purified subpopulations of B lineage cells from human adult (rib) bone marrow by cell sorting and panning. Limiting dilution analysis was then used for a clonal analysis of cells able to secrete IgG, IgA, or IgM spontaneously or after infection with EBV. Nonproliferating, high rate IgG or IgA producers occurred at frequencies of about one per 1000 marrow mononuclear cells. Their frequency and Ig production was unaffected by EBV, and they appeared not to express EBNA after exposure to EBV. These cells were Ia+, B1+, and over 85% expressed sIg of the IgM/D (up to 75%) and/or IgG/A isotypes (40 to 60%). B cells committed to the secretion of IgM represent 2 to 10% of marrow B lymphocytes. They were found to be Ia+/B1+/B2+/CALLA- and C3b receptor (CR3)-cells, and most (greater than 90%) required infection with EBV and proliferation to develop into IgM-producing lymphocytes. Thirty to 40% of these cells did not express Ig (H or L chain) on their surface, and therefore resembled pre-B cells at the beginning of the 4- to 5-wk culture period. Proliferating pre-B cells from adult human marrow have been described, but their conversion into IgM-producing cells has not been formally demonstrated. Although EBV induces IgM production, the expression of EBNA, and several rounds of cell division in these cells, the induction of stable (greater than 5 wk) growth transformation represents a rare event in these pre-B cells: in several thousand limiting dilution wells, not a single culture of sIg-cells showed stable growth transformation. The dichotomy between EBV-induced high-rate IgM responses and absent growth transformation discriminates activation and transformation as distinct aspects of EBV-induced B cell "responses", and suggests that cellular properties play critical roles for viral transformation. We propose a model in which cellular target genes for transforming sequences in the EBV genome are transiently expressed during B cell differentiation.

Separation of lymphoid and myeloid blasts in the mixed blast crisis of chronic myelogenous leukemia: no evidence for Ig gene rearrangement in CALLA-positive blasts.

Recent studies suggest that lymphoid blast crisis cells of chronic myelogenous leukemia (CML) expressing the common acute lymphoblastic leukemia antigen (CALLA) are B precursor cells, based on the demonstration of immunoglobulin (Ig) gene rearrangement similar to common acute lymphocytic leukemia. There is little evidence to suggest whether the cells with similar lymphoid characteristics in the mixed blast crisis of CML are also committed to B cell lineage. A patient in "mixed" blast crisis of CML was studied. On the basis of morphology, cytochemistry, and immunological studies, the blasts were classified as having either lymphoid or myeloid characteristics. A proportion of the leukemic blasts expressed CALLA, whereas others expressed My7 antigen. In order to characterize both populations of cell further, CALLA+ blasts and My7+ (myeloid) blasts were isolated by fluorescence-activated cell sorting. The My7+ cells were highly proliferative in cell culture blast colony assays, retained the Ph1 chromosome, and were indistinguishable from acute myelogenous leukemia blasts. The CALLA+ cells were also Ph1-chromosome positive, but in contrast, were poorly proliferative in vitro. Of particular note was their retention of germline configuration of Ig genes, thus distinguishing them from blasts in the lymphoid crisis of CML. We conclude that the lymphoid component in mixed blast crisis may represent a stage of differentiation prior to commitment to B lineage.

Quantitative expression of epitopes defined by murine monoclonal antibodies on DR molecules from different HLA haplotypes.

Monoclonal antibodies 50D6 and 21r5, reactive with human class II molecules, were analyzed quantitatively by flow cytometry and cellular radioimmunoassay for their binding to cells of different HLA-DR types. Monoclonal antibody 50D6 bound equally to cells of all DR types tested except DR7, where no reactivity was observed. Monoclonal antibody 21r5 was reactive with all cells. However, the percentage of DR molecules at the cell surface expressing 21r5 epitope varied with the DR type and increased as follows: DR3 = DR7 less than DR2 less than DR5 less than DR4 less than DR1. MAb 50D6 reacted with an epitope spatially related to but distinct from the 21w4 epitope present on all DR molecules. The 50D6 epitope was shown to be present on isolated DR1 molecules.

A flow cytometric method for the detection of adenosine deaminase in mononuclear cells.

The purpose of this study was to develop a flow cytometric method for the detection of adenosine deaminase (ADA) in a single cell suspension of mononuclear cells. Anti-human ADA antibody was purified by affinity chromatography on a column of Sepharose 4B to which calf ADA was covalently linked. This antibody was used for indirect immunofluorescent staining of cells fixed in 4% paraformaldehyde. The specificity of staining was proved by substitution of anti-human ADA with normal rabbit IgG and by absorption experiments. The fluorescence profile of the cells was then analyzed by flow cytometry. Two groups of cells were studied: (a) thymocytes, tonsil cells and peripheral blood mononuclear cells (PBMC), (b) ADA-positive and ADA-deficient cell lines. In each of these populations of cells a peak of specific immunofluorescence staining for the enzyme could be easily distinguished from weak background staining of control preparations. Within each group, the cell population with higher ADA activity also displayed a greater intensity of cell fluorescence. Flow cytometry provides a means for quantitation of ADA in individual mononuclear cells.