EA Rosette Research Articles

Lymphocyte subpopulations in cerebrospinal fluid and peripheral blood in multiple sclerosis.

Lymphocytes subpopulations in cerebro-spinal fluid (CSF) and peripheral blood (PB) from multiple sclerosis (MS) patients were studied. PB of MS patients contains the same prevalence of E and EA rosette forming cells compared with controls, consisting of patients affected by various "nonimmunological" neuropsychiatric diseases. Cytochemical identification by the method of acid esterases in PB demonstrated in MS a prevalence of lymphocyte subpopulations similar to controls, and a relatively high percentage of macrophages compared with other methods, especially in MS patients: this may partially account for variable results obtained by various authors with the rosette technique. In CSF a significant decrease of total T, and particularly of T gamma cells, was found. Since T gamma lymphocytes have a suppressor effect on B cell proliferation and Ig synthesis, their decrease could be related with Ig hypersynthesis commonly found in the central nervous system of MS patients.

(18) Lichen aureus

The clinical course of a patient with acute lymphoid immunoblastic leukaemia and prominent nodular haemorrhagic skin lesions is described. Cytological, cytochemical and electronmicroscopic studies were performed on bone marrow and skin blast cells. The absence of surface immunoglobulins and of the other markers for B lymphocytes (EA and EAC rosettes) and the presence of 30% of spontaneous sheep erythrocyte rosettes excluding an acute leukaemia with Burkitt's tumour cells, suggest that T cells are involved. Complete haematological and cutaneous remission was obtained with prednisone therapy.

DNA RELEASED FROM ISCHEMIC AND REJECTING ORGANS IS AN INDICATOR OF GRAFT CELLULAR DAMAGE

A27 Warm and cold ischemia are injurious for endothelial and parenchymal cells of the transplanted organ. “Passenger ” leucocytes and fragments of disintegrated endothelial cells are washed out from the transplant during warm revascularization period and disseminate in the recipient. Donor cellular debris contains fragments with genetic material. The question arises whether the amount of donor graft released DNA could be a measure of its damage caused by ischemia, preservation and rejection. Aim: To measure concentration of donor DNA in recipient blood, lymph nodes and spleen at various time points after heart transplantation. Methods: Male LEW (RT1l) and BN (RT1n) rats served as donors and female LEW (RT1l) as recipients. After tx, the recipient blood (B), lymph nodes (LN) and spleen (SPL) were harvested. Dendritic cells were obtained from recipient spleen after digestion, gradient separation and enrichment by EA rosetting. Genomic DNA was isolated and real time PCR with rat SRY specific primers was performed. Results: Retrieval of male DNA after 1h of PBL injection into female recipient was dose-dependent with highest accumulation in LN. The male heart wash out prior to tx after 1h ex vivo ischemia did not show any presence of DNA. 1h after tx in an allogeneic combination donor DNA was found at highest concentrations in B and some in SPL, 24h later the concentration in B was slightly lower but it became detectable in LN. 72h after tx DNA concentration in B and LN decreased in favor of SPL. 1 week after tx donor DNA was found only in SPL. In a syngeneic combination 24 h after transplantation donor DNA could be detected only in B, 72 h later only in SPL. After 1 week donor DNA reappeared in B, was present in LN reaching high values in SPL. Donor DNA was found in SPL dendritic cells after allogeneic but not syngeneic grafting, however, was present in macrophages in both combinations. Conclusions: Donor heart passenger cells do not contribute to DNA disseminated in recipient. Immediately and one day after transplantation, donor DNA is present mostly in B. Later it accumulates in SPL. Recipient blood donor DNA levels on days 1 to 3 provide insight into the degree of ischemic and post ischemic damage of the graft. Later they reflect the degree of rejection damage. High levels of donor DNA and methodological simplicity of DNA measuring justify works on a clinical test for evaluation of graft damage during its procurement for grafting.

Inhibition of Fcγ Receptors in the Plasma of Subjects with Down's Syndrome

Subjects with Down's syndrome have several immunological abnormalities. We examined the sera of 29 subjects with Down's syndrome for the presence of Fcγ receptor blocking and for the presence of anti-ssDNA antibodies by EA rosette inhibition. Fifty-five percent of Down subjects had levels of inhibition above the upper limit of normality in comparison to 7% of normal controls. The finding that after polyethylene glycol precipitation of selected sera giving high levels of EA rosette inhibition there was a reduction or a disappearance of the EA rosette inhibition could indicate that the blocking factors detected behaved as immune complexes. Since almost all subjects with anti-ssDNA antibodies also had elevated values of EA rosette inhibition, a role for immune complexes eventually formed with autoantibodies in an Fc-mediated immunoregulatory system is suggested.

Detection of lymphocyte Fcγ receptor-blocking factors by the EA rosette inhibition assay: Refinement of the conventional method and development of a novel flow-cytometric assay

Serum factors which interact with human peripheral blood lymphocyte Fcγ receptors (FcγRs) may be detected in vitro by the EA rosette inhibition assay (EARIA). This assay has been used to detect circulating immune complexes and certain alloantibodies directed against cell surface antigens situated in close proximity to FcγRs. Three main types of FcR-blocking factor have been demonstrated by the EARIA in human serum following exposure to alloantigens. A strong correlation was observed between the presence of one of these FcR-blocking factors (FcBF1) and human renal allograft survival. This factor was previously shown to bind preferentially to CD32 + B cells and to inhibit antibody synthesis. In this study we have shown that detection of FcBF1 by the EARIA depends on the type of erythrocyte and on the amount of antibody used to sensitise the erythrocytes. Furthermore, we have developed a flow-cytometric version of the EARIA which is rapid, reproducible and, most importantly, objective. Inter-laboratory comparisons using this standardised EARIA should now be possible.

Possible roles of LFA-1 and Fc gamma receptors on the functional immaturities of cord blood polymorphonuclear leukocytes.

The functional immaturity of PMNs is one of the major causes of overwhelming sepsis in newborns. In this study, we observed functions and surface markers of PMNs to investigate what causes the functional immaturity of PMNs in newborns. As results, the percentage of EA rosette forming PMNs (58.5 +/- 15.5%) and the chemotactic movement (0.14 +/- 0.09 mm) of cord blood PMNs were significantly lower than those of adult peripheral blood PMNs (70.8 +/- 9.9%, 0.60 +/- 0.34 mm). Cord blood PMNs showed decreased glass adherence and ADCC activity. The expression of Fc gamma RII or Fc gamma RIII was a little lower than those of adult peripheral blood PMNs, but the expression of Fc gamma RI (43.1 +/- 26.8%) was significantly higher than that of adult peripheral blood PMNs (3.2 +/- 1.8%). There was a significant difference in LFA-1 expression between EA rosette forming PMNs (92.9 +/- 9.1%) and EA rosette non-forming PMNs (25.6 +/- 22.6%). From these results, it is assumed that neonatal PMNs may consist of heterogeneous populations. And the relatively high percentage of EA rosette non-forming PMNs which express a low level of LFA-1 may be responsible for the functional immaturity of cord blood PMNs.

CD16 on human γδ T lymphocytes: Expression, function, and specificity for mouse IgG isotypes

We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human γδ T cells. CD16 is expressed by the majority of γδ T cells in peripheral blood and by part of the γδ T cell clones. The amount of CD16 expressed on γδ T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated γδ T cells. Like CD16 on CD3 −CD16 + natural killer (NK) cells, CD16 on γδ T cells can act as an activation site triggering cytotoxic activity. CD16 + γδ T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of γδ T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of FcγR + target cells by CD16 + γδ T cell clones. The mouse IgG-isotype specificity of CD16 on γδ T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3 −CD16 + NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both γδ T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.

Properties of Nine Continuous B‐Cell Lines Established from Enzootic Bovine Leukosis Tumors

We established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)-positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg-positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B-lymphocytes. Morphological observation also revealed that they had the morphology of B-lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C-143, which recognizes the tumor-associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B-lymphocytes of EBL.

Immunomodulating Effects of Various Phenothiazines and Related Tricyclic Compounds in Vitro and in Vivo

A biotechnological approach to the immunomodulating ability of various phenothiazines and related tricyclic compounds in vitro and in vivo was made. The effect of these compounds was studied on various mononuclear leukocyte functions such as antibody-dependent cell-mediated cytotoxicity, blast transformation of lymphocytes by three mitogens, phytohemagglutinin, concanavalin A and pokeweed mitogen (PWM), rosette formation with erythrocytes and with antibody coated erythrocytes (EA), as well as on polymorphonuclear leukocyte functions including phagocytosis and chemiluminescence emission. Moreover, the ability of these compounds to modulate the bioluminescence emission capacity of Escherichia coli cloned with lux genes was measured. Also in vivo studies were made by measuring the number of haemolytic plaque forming cells in the spleen of mice after drug administration. The electrochemical structure of these tricyclic compounds was shown to be in good correlation with their immunomodulating activity on all the functions except EA rosette formation and on blast transformation by PWM showing the role of charge transfer complex formation in the drug-target cell interaction. These results suggest that the biological effectiveness of phenothiazines and related tricyclic compounds can be presumed from their electrochemical properties and a tricyclic compound with a given biological effect can be theoretically designed.

Novel Cell Surface Antigens Expressed on Mouse Alveolar Macrophages

Two new cell surface antigens expressed on mouse alveolar macrophages were defined by rat monoclonal antibodies. One marker, AVM-1, was detected on mouse alveolar macrophages, but it was undetectable on resident peritoneal cells, thioglycollate medium-induced peritoneal cells, and splenic macrophages. Splenic lymphocytes, thymocytes and bone marrow cells were also AVM-1 negative. Anti-AVM-1 monoclonal antibody immunoprecipitated a single polypeptide with a molecular weight of 200,000. Of particular interest was the finding that the anti-AVM-1 antibody could inhibit the formation of EA and EAC rosette on macrophage line cells. A second antigen (AVM-2) was also present on alveolar macrophages, and its molecular weight was 38,000.

Modulation of type II Fcγ receptor expression on activated human B lymphocytes

We have monitored Fc gamma RII expression during the activation of human B lymphocytes by simultaneous analysis of monoclonal antibody (mAb) binding and EA rosetting. The expression of Fc gamma RII showed a biphasic time course. Initially, a transient increase of Fc gamma RII with no ligand-binding capacity was observed with mAb staining as early as 10 min after stimulation by the F(ab')2 fragment of anti-human IgM or phorbol 12-myristate 13-acetate and then after 3 to 24 h a decrease in the number of Fc gamma RII+ cells was seen. Trypsin-like serine protease activity also appeared in the lysate of activated B cells at this time. On the 2nd day of activation a significant enhancement of Fc gamma RII expression was observed, mainly on enlarged blast cells as monitored by both mAb and by ligand binding (EA rosette). At the same time, soluble fragments of Fc gamma RII with the ability to bind human Fc were detected in the supernatant of activated B cells, probably as a result of proteolytic cleavage. These findings suggest that activated B cells are identical with the population of mononuclear cells which shed Fc gamma R when incubated at 37 degrees C. The ability of activated but not resting B cells to release Fc gamma RII correlates with the expression of early activation markers and with the appearance of a trypsin-like serine protease activity of the same cells; thus, the release of Fc gamma RII occurs in the early G1 phase of cell cycle as a result of proteolysis. Later the release of Fc gamma RII is accompanied by the enhancement of Fc gamma RII expression before the cells reach the S phase. The fragments of cleaved Fc gamma RII had an apparent molecular mass of 33 and 14-18 kDa under nonreducing conditions, and upon reduction fragments of smaller size were observed.

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.

A flow cytometric rosetting assay for the analysis of IgG-Fc receptor interactions

We have developed a sensitive and flexible method for the qualitative evaluation of IgG-Fc receptor interactions in cell suspensions. The assay is based on the flow cytometric quantitation of antibody-coated erythrocyte (EA) rosetting using fluorescein-labelled indicator erythrocytes (E). The number of IgG molecules on indicator E, an important parameter in EA rosetting, was estimated by calibrated flow cytometry. EA binding quantitated by this method was correlated with microscopically evaluated rosette formation. Besides automated quantitation of EA binding, this method offers the additional advantage of simultaneously using a second fluorescence parameter, permitting analysis of FcR activity in subpopulations of cells. As an example of the applicability of this approach the binding characteristics of E sensitized with a series of murine heavy chain isotype switch variant monoclonal antibodies against glycophorin A, to the low affinity receptor on K562 cells were determined. Remarkably, the results suggest a comparable affinity of FcγRII on these cells for immunoglobulins of the murine IgG1, IgG2a and IgG2b isotypes.

Immune complex-mediated inhibition of lymphocyte Fc-γ receptors in the plasma of patients with type 1 (insulin-dependent) diabetes mellitus: Association with anti-ssDNA antibodies

Sera of 43 patients with type 1 (insulin-dependent) diabetes and from 42 normal subjects were examined for the presence of Fc-γ receptor-blocking IgG immune complexes, detected by EA rosette inhibition, and for the presence of anti-ssDNA antibodies by ELISA. Forty-four percent of diabetic patients had levels of inhibition above the upper limit of normality in comparison to 7% of normal controls. Anti-ssDNA antibodies were found in the sera of 14 out of 43 diabetic patients. Ten out of 14 anti-ssDNA positive patients (71.5%) had inhibition levels above the upper limit of normality in comparison to 9 out of 29 (31%) of the anti-ssDNA negative population. The difference was statistically significant ( P < 0.005). Levels of EA rosette inhibition were found to be high in patients with duration of diabetes of less than 2 years and correlated with high prevalence of anti-ssDNA antibodies. The percentage of EA rosette inhibition was found not to correlate with the levels of Clq-binding immune complexes, suggesting that immune complexes detected by EA rosette inhibition may belong to a pool of noncomplement-fixing immune complexes. The possible role of immune complexes with autoantibodies anti-ssDNA in the mechanism triggering and perpetuating autoimmune phenomena in diabetes is discussed.

High‐Dose, pulse intravenous methylprednisolone enhances fcγ receptor–mediated mononuclear phagocyte function in systemic lupus erythematosus

The benefit of high-dose, pulse intravenous methylprednisolone (IVMP) for some patients with active lupus nephritis would appear paradoxical, since active nephritis is associated with profound abnormalities in Fc gamma receptor function, and several studies have demonstrated that glucocorticoids decrease monocyte Fc gamma receptor expression and phagocytic function. To resolve this paradox, we investigated the possibility that pulse IVMP might enhance monocyte Fc gamma receptor function in patients with systemic lupus erythematosus (SLE). Circulating immune complex (CIC) levels, Fc gamma receptor-mediated clearance, and Fc gamma receptor-dependent monocyte function were analyzed in 23 SLE patients before and after pulse IVMP (1 gm daily for 3 days). A biphasic response in CIC levels, determined by a staphylococcal protein A binding assay, was observed. Initially, CIC levels increased within 2-4 hours after the first dose of pulse IVMP and then decreased by 50% within 24-48 hours after the completion of therapy. Fc gamma receptor-mediated binding and phagocytosis of IgG-sensitized erythrocytes (EA) by monocytes in vitro were significantly enhanced 24 hours after the final dose of pulse IVMP (pre-IVMP versus post-IVMP 43 +/- 14% versus 53 +/- 12% EA rosettes, P less than 0.01; 3.00 +/- 1.04 versus 3.99 +/- 1.30 EA ingested/monocyte, P less than 0.01). In contrast, there was no change in the phagocytosis of an Fc gamma receptor-independent probe, neuraminidase-treated erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

T and B lymphocytes in horses persistently infected with equine infectious anaemia virus.

The percentage of T and B lymphocytes in the peripheral blood of horses chronically infected with equine infectious anaemia (EIA) virus was determined and the results were compared with the percentage of these cells in healthy uninfected horses. Cells with membrane receptors for sheep erythrocytes (T and active T lymphocytes) were determined by E and A rosette techniques, while cells with receptors for the C3b component of complement and those with receptors for mouse erythrocytes (B lymphocytes), were determined by the EAC rosette method. The percentage of Fe positive cells was assayed by the EA rosette test. The majority of peripheral blood lymphocytes (PBL) from both uninfected and EIA-infected horses formed rosettes of each kind with only three erythrocytes indicating a low density of the corresponding receptors on the cell membrane under the condition of the assays used. The percentage of T lymphocytes in the peripheral blood of diseased horses (52.4 +/- 1.6%), as detected by E rosettes, was significantly (p less than 0.01) higher than in control animals (42.4 +/- 3.5%). In clinically healthy horses 8.9 +/- 1.1% of PBL were identified by A rosettes as active T cells, whereas animals with a chronic form of EIA had a much lower (p less than 0.001) percentage of these cells (4.7 +/- 0.7%). In the B lymphocyte subpopulations the percentages of cells bearing Fc and C3b receptors were markedly elevated (p less than 0.001) in EIA-infected horses (24.7 +/- 0.8% and 42.8 +/- 2.2% respectively) as compared to uninfected animals (15.1 +/- 1.4% and 29.6 +/- 1.2% respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Retinoic acid regulates IgG Fc receptor expression in human myelomonocytic leukemia cells and normal peripheral monocytes.

The regulation of IgG Fc receptor (Fc gamma R) expression by retinoic acid (RA) in human myelomonocytic cells at different stages of maturation was studied. RA suppressed IgG-coated erythrocyte (EA) rosette formation of myelomonocytic cells blocked at relatively late stages of differentiation such as ML-1, U-937, THP-1-T, normal monocytes, and fresh cells of patients with acute myelomonocytic leukemia. However, RA increased the percentage of EA rosetting promyelocytes of HL-60 and of patients with acute promyelocytic leukemia and a part of myeloblasts isolated from acute myelogenous leukemia patients. Other myeloblasts including KG-1a, KG-1, and fresh cells from patients with acute myelogenous leukemia were not affected. A kinetic study using HL-60 and THP-1-T demonstrated that an increase required at least a 48-h exposure and that the maximum decrease required approximately 6 h. The RA effect on both cell lines was dose-dependent. The number of Fc gamma R of HL-60 and THP-1-T treated with RA became very close, although untreated THP-1 had almost 10 times as many as HL-60. Kd for IgG in both THP-1-T and HL-60, either untreated or treated with RA, remained unchanged. These observations indicate that one of the important roles of RA is regulation of Fc gamma R expression in myeloid cells.

129: MONOCYTE (Mo) FUNCTION IN CHILDREN WITH JUVENILE RHEUMATOID ARTHRITIS (JRA)

A sample of 20 children with juvenile rheumatoid arthritis and 23 matched controls were studied in an attempt to verify null hypothesis that Mo function in children with JRA did not differ from that observed among healthy controls. The suppressor activity of Mo was studied by inhibition of 3H-thymidine incorporation in coculture with control PBMC stimulated with PHA. Functional expression of Fc receptor (FcR) was determined by EA rosettes, whereas expression of MHC class II determinants was assesed with HAK-75 and 14-4-4s mAb. It was shown that patients' Mo provided no appreciable suppression of PBMC stimulated with PHA. A significant (p <0.05) decrease of Mo (FcR+) in patients blood was observed. The number of Mo revealing HLA-DR determinants reacting with HAK-75 mAb was similar in both groups of children. However, the population of Mo with Ia. 7 receptor identified by 14-4-4s mAb was significantly (p < 0.05) diminished among the patients. A decreased Mo suppressor activity combined with decreased expressivity of Fc and Ia. 7 receptors justify the rejection of null hypothesis and imply an abnormal regulatory function of Mo in children with JRA.

Fc receptor for shark IgM

Fc receptors for shark IgM have been demonstrated on shark leukocytes. Measurement of receptor binding required treatment of leukocytes with Cytochalasin D to inhibit phagocytosis. EA rosetting assays were carried out using human erythrocytes coated with shark anti-human antibody. Binding to shark leukocytes was demonstrated to be specific to shark IgM in that affinity purified shark IgM and purified Fc5μ fragments could block rosette formation, but not shark transferrin, bovine serum albumin or fetal bovine serum. The binding was shown to be saturable and reversible, characteristic of receptor-ligand interaction. Further, it was shown that affinity purified, radioiodinated IgM could also bind Cytochalasin D-treated shark leukocytes in a manner analogous to rosetting. We conclude that Fc receptors appeared early in evolution, and that previous difficulties in demonstrating the Fcμ receptor resulted from non-specific binding associated with phagocytosis.

Comparative study of alloimmune reactions induced by leukocyte and platelet transfusions in humans: characteristic changes of activation markers, gamma interferon, and FcR blocking antibody production

This study was undertaken to define immune responses induced by leukocyte and platelet transfusions. We offer evidence that activation by alloimmunization with intravenously administered “buffy coat” cells results in immune alterations similar to the early rejection episodes following kidney transplantation. Thus, IL-2 and HLA-DR expression increased on PBL 2 and 3 days after antigen exposure and paralleled elevations of IFN-γ, neopterin, and beta 2 microglobulin levels. No significant changes were detected in the number of T-cell subpopulations. Alloimmunization by purified platelets alone that express only class I MHC antigens failed to induce the above alteration, even after repeated exposure. Repeated alloimmunization with “buffy coat” cells or isolated platelets stimulated a progressive increase up to 100% blocking activity on EA rosette formation. We conclude that the expression of activation markers on PBL and the production of IFN gamma and neopterin reflect the early phase of alloimmune response induced by leukocytes. This type of alloactivation is not a prerequisite for the development of FcR-blocking antibody, which is produced after pure platelet transfusion as well.