Influence of aluminium on the immune system--an experimental study on volunteers.

The purpose of this study was to examine whether oral exposure to aluminum (Al) can affect the human immune system. Eighteen healthy volunteers (mean age 42, 28-57 yr) were divided into a test group (9 females, 4 males) and a referent group (3 females, 2 males). Over 6 weeks, the test subjects ingested 10 ml of antacid (aluminum hydroxide, 59 mg Al/ml) three times daily. Aluminum was analyzed in urine before and during the exposure period (ICP-MS). Blood samples were used for analysis of lymphocyte subpopulations, mitogen-induced lymphocyte proliferation and in vitro production and circulating plasma concentrations of immunoglobulin (Ig) A, IgG, IgM, interleukin (IL) -2 and IL-4. Urinary Al concentration in the test subjects was approximately 10- to 20-fold higher than in the referent group during exposure. This indicates that ingestion of an Al-containing antacid is associated with an Al absorption far above that originating from food and drinking water. In both referents and test subjects the lymphocyte subpopulations, lymphocyte proliferation and the in vitro Ig and IL production showed similar, time-dependent changes before as well as during the exposure period. No major differences were seen between the referent and test groups regarding the immune parameters, except for a slightly smaller CD8+CD45R0+ population (primed cytotoxic T-cells), in the exposed individuals as compared to the referents. The results also show that subjects on antacid therapy may constitute a suitable population for studying biological effects of high-dose oral exposure to Al.

Ex vivo PKH26-labelling of lymphocytes for studies of cell migration in vivo.

A prerequisite for studies of cell migration is that the cells of interest can be appropriately labelled and subsequently easily traced. The use of radioisotopes or fluorescent substances that bind covalently to the cell surface, e.g. fluorescein isothiocyanate (FITC) or rhodamine isothiocyanate (RITC), have limitations such as rapid loss of the labelling, toxicity and interference with cell surface molecules. In the present work the authors labelled rat spleen lymphocytes with the fluorescent labelling molecule PKH26, which is incorporated into the lipid bilayer of cytoplasmic membranes. The labelled lymphocytes were injected intravenously into syngeneic recipients and 2 or 6 days later the lymphocytes were detected in various organs by using flow cytometry and fluorescence microscopy. As could be expected, the lymphocytes homed to lymphoid tissues, preferably the spleen, and no labelled cells were found in non-lymphoid organs such as the heart and the kidney. Membrane labelling proved to be intense, uniform and stable and PKH26 positive cells were easily detectable in fractions less than 0.2% in peripheral blood and the various tissues after 6 days of in vivo circulation. Thus, the PKH26 dye appears to be suitable for labelling cell populations used in the study of cell migration in vivo, both under normal conditions and when specific immunological processes are taking place, such as graft rejection and tumour growth.

Phenotype transition of CD4+ T cells from CD45RA to CD45R0 is accompanied by cell activation and proliferation.

An investigation of proliferation and activation events in subsets of human CD4+ cells, defined by their expression of CD45RA and CD45R0, is reported. A single-laser based assay for the study of multiple surface antigens and two-parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4+CD45RA+ CD45R0-, CD4+CD45RA-CD45R0+ subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with flow cytometry on the basis of light-scattering properties and staining with anti-CD45RA, anti-CD45R0, and anti-CD4 markers. Sorted cells were double stained with anti-BrdUrd-antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4+ CD45RA+CD45R0- and CD4+CD45RA-CD45R0+. After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4+CD45RA-CD45R0+ population. These findings were further corroborated by four-colour staining with anti-CD4, anti-CD45RA, anti-CD45R0, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA-DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L-selectin) in each of the different subsets was also investigated. All the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4+ CD45RA-CD45R0+ cells than the CD4+ CD45RA+CD45R0- cells, indicating a subpopulation of CD4+CD45RA-CD45R0+ cells responding more slowly to the mitogenic stimulation.

Multicolor flow cytometric analysis of the CD45 antigen provides improved lymphoid cell discrimination in bone marrow and tissue biopsies.

Samples from bone marrow or non-hematopoietic tissue such as solid organ biopsies often contain an excess of non-leukocytes exhibiting lymphocyte-like light scatter characteristics, making it sometimes difficult to define satisfactory light scatter lymphocyte gates. To circumvent this, we describe here a multiparametric method of identifying lymphoid cells by expression of the CD45 antigen, in conjunction with light scatter parameters. A 'third color'-conjugated anti-CD45 antibody was included with every FITC/PE double staining, thereby permitting live or list mode analysis gating on CD45 positive cells. The triple-staining technique was applied to (a) human bone marrow, showing that special attention has to be given to the enumeration of B cells, and (b) to liver biopsies, where gating on CD45 fluorescence and orthogonal light scatter was shown to clearly resolve all lymphocyte subsets from debris. All cell types examined in tissue biopsies as well as T and NK cells in bone marrow were best distinguished by gating on bright CD45 expression in conjunction with low orthogonal light scatter, while accurate identification of marrow B cells relied upon including all levels of CD45 intensity. The multicolor gating procedure, aimed mainly at immune-monitoring of non-malignant tissues, is applicable to most kinds of single cell samples, and may prove to be an aid for lymphocyte gating in cases where leukocyte populations are not clearly resolved on a light scatter basis alone.

Efficient killing of chronic B-lymphocytic leukemia cells by superantigen-directed T cells.

In vitro studies have indicated that chronic lymphocytic leukemia of B-cell origin (B-CLL) is resistant to cytotoxic effector lymphocytes such as natural killer and lymphokine activated killer (LAK) cells. We show here that B-cell cells are sensitive to Staphylococcal enterotoxin (SE) A-directed T-cell killing. Activation of the target cells by phorbol ester (tetradecanoyl phorbol acetate, [TPA]) greatly enhances their sensitivity to lysis. In SE-dependent cellular cytotoxicity (SDCC), members of the SE superantigen family form a bridge between T cells and target cells expressing major histocompatability complex class II molecules. Binding of SEA to the T-cell-receptor V beta region induces a strong cytotoxic capacity and cytokine production. Cells from 9 B-CLL patients were cultured in the presence or absence of TPA and used as targets in a 4-hour SDCC assay using an allogeneic T-cell line as effector. At an effector:target cell ratio 30:1, 70% to 80% of TPA-induced B-CLL cells were killed. Even at the effector:target ratio of 3:1, 47% +/- 6% of TPA-activated B-cell cells were lysed compared with 13% +/- 2% of resting cells (P < .001). A T-cell line established from a B-CLL patient killed autologous tumor cells as efficiently as allogeneic effectors. SEA-directed T cells were far more lytic to B-CLL cells compared with LAK cells or lectin (phytohemagglutinin-directed T cells. Mechanisms of SDCC lysis were investigated. Effector plus target cell supernatants contained high levels of tumor necrosis factor (TNF)-alpha and interferon-gamma, but these supernatants were not directly toxic to B-CLL cells in short term culture. High concentrations of recombinant TNF-alpha or TNF-beta had no lytic effect. Addition of neutralizing anti-TNF-alpha and anti-TNF-beta antibodies into the SDCC assay did not inhibit SEA-directed T-cell killing. TPA-activated B-CLL cells showed a 1.2- to 13-fold increased expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA)-1, and LFA-3, whereas expression of HLA class II molecules increased up to 5 times. The expression of CD72, CD40, and BB-1/B7 increased 1.8 to 4.5 times. The role of these surface molecules in SDCC was analyzed in blocking experiments with monoclonal antibodies. Antibodies to ICAM-1, CD18, and HLA-DR abolished the cytotoxicity, and a substantial reduction was seen with antibody to CD72.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential membrane labelling of human lymphocyte subsets by PKH-2 examined by multiparameter flow cytometry. A possible correlation between lipid composition of cellular membranes and functional properties?

Human peripheral blood lymphocytes were stained simultaneously with the lipophilic membrane label PKH-2 and surface markers and analysed by multiparameter flow cytometry. A biphasic staining distribution was observed for T (CD3+) as well as B (CD19+) cells. Within the T subset, CD4+ cells stained almost exclusively with bright PKH-2 intensity, while CD8+ cells exhibited a more pronounced bimodal staining pattern. No difference in staining intensity was noted for CD4+CD45R0+ vs. CD4+CD45R0- cells. This multiparameter staining method demonstrates differences in membrane lipid composition between different human lymphocyte subsets, and suggest that caution must be exercised when evaluating PKH-2 labelling of human lymphocytes.

Studies of fibrinogen binding to platelets by flow cytometry: an improved method for studies of platelet activation.

Platelet function is dependent upon membrane receptors and their interaction with other proteins. Platelet activation appears to cause a structural change of the glycoprotein IIb/IIIa complex that exposes the fibrinogen binding site, which subsequently binds fibrinogen. Fluorescence-activated flow cytometry (FACS) is an efficient method for studying membrane proteins. Flow cytometry gives single-cell data, allowing the detection of only a small proportion of labelled platelets in whole blood without any washing steps. One problem with this method is that the labelled antibodies and the antigen, if present in plasma, form an immune complex, which may cause false positive reactions due to interaction between mammalian IgG and Fc gamma receptors on the platelets. We show that immune complexes with chicken IgG do not activate human platelets. We have developed a method for measuring platelet-bound fibrinogen in whole blood and platelet-rich plasma utilising fluorescein isothiocyanate (FITC)-conjugated chicken antibodies directed towards human fibrinogen. As low as 1% activated platelets could be detected without interference from Fc-interactions.

Blood and liver-infiltrating lymphocytes in primary biliary cirrhosis: increase in activated T and natural killer cells and recruitment of primed memory T cells.

We used two-color and three-color flow cytometric analysis to study phenotypical activation and functional subsets of T and natural killer cells in the blood and liver tissue of patients with primary biliary cirrhosis, other chronic liver diseases and the blood of healthy subjects. The changes in blood lymphocyte phenotype in patients with primary biliary cirrhosis and other chronic liver diseases were similar and comprised elevated relative or absolute numbers of activated human leukocyte antigen-DR + T subset (CD4+ and CD8+) cells and DR+ natural killer-like (CD16+) cells. B cell (CD19+) numbers were normal. In primary biliary cirrhosis a selective reduction in T cells of suppressor-inducer (CD45RA + CD4 + ) type was registered. The human leukocyte antigen-DR expression among CD4+ T cell subsets was investigated further in primary biliary cirrhosis and healthy controls using triple antibody flow cytometric analysis. Phenotypical cell activation was confined to helper T cells of the primed, memory (CD45RO + CD4+) type. The decrease in suppressor-inducer T cells in primary biliary cirrhosis was paralleled by a reciprocal increase in primed memory T cells. Several significant differences were observed when blood and liver-infiltrating cells from primary biliary cirrhosis patients were compared. In the liver tissue, the CD4/CD8 ratio was decreased, the relative activation of T-subset cells and NK cells was further increased, the suppressor-inducer T subset was further depressed and the primed memory T subset was increased. The cytotoxic T-cell subset (CD11b-) dominated within the CD8+ population. In liver tissue from other chronic liver disease subjects, a lower CD4/CD8 ratio was found compared with primary biliary cirrhosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Single laser flow cytometric detection of lymphocytes binding three antibodies labelled with fluorescein, phycoerythrin and a novel tandem fluorochrome conjugate.

In order to implement three-color surface immunofluorescence on a single laser flow cytometer, we combined a new fluorescent tandem conjugate (TC, R-phycoerythrin plus Texas red, available as DuoCHROME) with fluorescein (FITC)- and phycoerythrin (PE)-labelled monoclonal antibodies for the simultaneous detection of three antigens on individual lymphoid cells. Considerable amounts of fluorescence leaked from the PE component of the TC complex into the PE detector, which necessitated compensation of the PE signal. Single and double stainings of peripheral lymphocytes with FITC-, PE- and/or TC-labelled antibodies revealed that the individual labels all identified populations of similar size, and that subsets of T cells could be properly and accurately detected with TC as one label. Triple staining was used to study phenotypically activated HLA-DR+ cells within functionally important subsets of CD4+ T cells, i.e., CD45R+ CD4+ (naive, 'suppressor inducer' T) and UCHL1+ CD4+ (memory, 'helper' T) cells. In the blood of healthy subjects as well as patients with primary biliary cirrhosis, CD4+ UCHL1+ (i.e., memory) T cells were significantly more activated than CD4+ UCHL1- cells. The reciprocal CD45R+ CD4+ (naive) T subset expressed HLA-DR to a much lesser extent. In patient samples, this difference in CD4+ T subset activation was more pronounced than among normal lymphocytes. The described triple immunofluorescence staining technique facilitates true multicolor analysis of individual cells on a single laser flow cytometer. Such multiparameter investigations may prove to be important for the further understanding of the phenotypic and functional diversity of immunocompetent cells.

Isoforms of leukocyte common antigen (CD45) on human neoplastic hematopoietic cell lines.

Leukocyte common antigen (LCA) isoform expression patterns were examined on different human hematopoietic cells and cell lines with the aid of the immunoblotting technique utilizing two anti-LCA monoclonal antibodies elicited by an ALL cell line and able to detect four isoforms (180, 195, 205 and 220 kDa) of LCA on peripheral blood lymphocytes from healthy donors and some neoplastic hematopoietic cell lines. Fourteen leukemia/lymphoma cell lines of different origin and immunophenotype, as well as three nonhematopoietic human tumor cell lines were examined by this technique. Human hematopoietic cell lines expressed two to four different polypeptide chains (180, 195, 205 and 220 kDa). Non-T, non-B cell lines, some B-cell lines and monocyte lymphoma cell line U 937 predominantly expressed higher molecular weight isoforms. In myeloid leukemia cell lines examined the expression of lower molecular weight isoform(s) predominated. Individual variability in LCA isoform patterns on cell lines precluded the strict correlation between these patterns and immunophenotype of the examined cell lines.

Regeneration of functional and activated NK and T sub-subset cells in the marrow and blood after autologous bone marrow transplantation: a prospective phenotypic study with 2/3-color FACS analysis.

The phenotypic reconstitution of lymphoid cells in the bone marrow and peripheral blood was examined prospectively in 27 patients who underwent autologous bone marrow transplantation (ABMT) for leukemia/lymphoma. Patterns of activation within NK and T cell subsets as well as T sub-subsets were studied with monoclonal antibodies in two- and three-color FACS analysis. NK-like cells (CD16+) were found to be increased in the peripheral blood and bone marrow after ABMT. The expression of two activation markers, 4F2 and HLA-DR, was sustainedly increased within this subset. High numbers of CD4+ and CD8+ T cells carrying surface HLA-DR were found early after ABMT both in blood and marrow. The T suppressor inducer cell sub-subset (CD45R+ CD4+) was severely depressed in both the blood and marrow 6-9 months post-ABMT. Using three-color FACS analysis, half of this T sub-subset was shown to express HLA-DR+. The levels of T suppressor effector-like cells (CD11+ CD8+) remained within the normal range in both peripheral blood and bone marrow during follow-up. The HLA-DR expression was elevated and equally distributed between the CD11- CD8+ and CD11+ CD8+ sub-subset cells. There was no major impact of marrow T cell purging or CMV carrier status on the phenotypic NK/T cell reconstitution. The present results provide an immune phenotypic basis for the suggested generation of anti-leukemic NK-like and T suppressor-like activity after ABMT.

B lymphocyte regeneration in marrow and blood after autologous bone marrow transplantation: increased numbers of B cells carrying activation and progression markers.

The reconstitution of B cells in the bone marrow and peripheral blood was prospectively studied in 27 patients undergoing autologous bone marrow transplantation (ABMT). No major differences in B cell regeneration patterns were recorded between patients receiving marrows purged of B cells (anti-CD10 + 19; n = 17) and patients receiving unpurged marrows (n = 10). Compared with healthy controls, elevated absolute and relative numbers of B cells were recorded in the blood and marrow at +6 and +12 months in both groups of patients. CD23+ B cells were severely depressed during the first three months post ABMT, indicating immaturity. A twofold increase in B cells carrying the activation marker 4F2 was recorded in the marrow at +1 month. Serum immunoglobulin levels (IgG, IgA, IgM) were within low-normal range throughout the study. The depressed B cell responses reported after allogeneic and autologous BMT could in part be explained by the low expression of the CD23 antigen on B cells after such therapy.

Monoclonal antibody directed to MHC class I antigen (Bra23/9): characterization and utilization for study of antigen expression in differentiation of U 937 cell line.

The monoclonal antibody Bra23/9 (IgG2a) elicited by a non-T, non-B ALL cell line (REH), reacted in microscopic immunofluorescence, enzyme-linked immunoassay (ELISA), complement-dependent cytotoxicity tests and immunocytofluorometric measurements with hemopoietic cell lines and peripheral blood lymphocytes, monocytes and granulocytes from healthy donors in a pattern characteristic for MHC class I antigens. Immunoprecipitation of lactoperoxidase radioiodinated cell surface proteins and surface sialoglycoproteins radiolabeled by sodium metaperiodate/tritiated sodium borohydride technique confirmed the structure gp44,p12 (consisting of a 44 kDa glycopeptide linked with a nonglycosylated 12 kDa peptide) typical for MHC class I antigen(s) as a structure recognized by the Bra23/9 monoclonal antibody. The increase of MHC class I antigen density (immunofluorescence intensity) induced by a phorbol ester (TPA) in monoblast U 937 lymphoma cell line was observed by immunocytofluorometry as a predominant tendency in several TPA-induction experiments, where a certain variability among individual experiments in TPA-induced MHC class I antigen alterations was observed.

Immunocytometric characteristics of a monoclonal antibody (Bra55) recognizing the leukocyte common antigen (LCA).

The immunofluorescence cytofluorometric reactivity pattern of monoclonal antibody Bra55 (IgG1) elicited with a non-T, non-B ALL cell line (REH), with a panel of human neoplastic hemopoietic cell lines (including non-T, non-B, T and myeloid leukemia cell lines) and with isolated peripheral blood lymphocytes, monocytes and granulocytes from healthy donors corresponded to the previously described microscopic immunofluorescence, ELISA, immunoprecipitation and immunocytochemic data indicating that this monoclonal antibody recognizes a 170-220 kDa cell surface glycoprotein (leukocyte common antigen) expressed selectively on hemopoietic cells. The purified, FITC-conjugated Bra55 monoclonal antibody was effectively inhibited in its binding to the surface of LCA-positive cells by reference anti-LCA monoclonal antibodies; no inhibition of this activity by LCA-unrelated monoclonal antibodies (such as anti-MHC class I and class II antibodies) was observed. These data confirm the previously reported hemopoietic cell specificity (anti-LCA, CD45) of the Bra55 monoclonal antibody.

Detection of triple antibody-binding lymphocytes in standard single laser flow cytometry using colloidal gold, fluorescein and phycoerythrin as labels.

Goat anti-mouse antibodies conjugated with colloidal 40 nm gold particles (G40) were used as secondary layers to stain human T lymphocytes in an attempt to extend the polarized light epi-illumination microscopic technique to flow cytometric multiparameter analysis. G40-labelled T cells were further stained with phycoerythrin (PE, red)- and fluorescein (FITC, green)-conjugated antibodies with specificity for the same cells. The cell samples were then analysed on a standard flow cytometer equipped with one laser operating at 488 nm. G40-labelled cells were detected in the side scatter (90 degrees) channel and fluorescent cells in the red and green fluorescence channels, respectively. Single labelling of the same T cell subsets with either G40-, FITC- or PE-conjugated antibodies yielded similar results, and cell mixture experiments did not show interference between gold and fluorescence labels. Triple staining experiments with three differently conjugated antibodies showed that subpopulations of cells were labelled in an independent manner with one, two or three antibodies in proportions expected from fluorescence controls. It was, therefore, possible to detect subset, sub-subset and activation markers on individual T cells. Our experiments show that immunogold staining of cell populations can be detected in routine one-laser flow cytometry. Further, the gold label can be combined to give two-color staining with ordinary red and green fluorochrome-conjugated antibodies thereby permitting rapid and precise triple antibody staining of individual cells. This methodology provides a powerful tool for the analysis of cellular antigenic diversity among, e.g., immunocompetent cells.