Rosette-forming Lymphocytes Research Articles

Influence of surgical treatment of periapical lesions on serum and blood levels of inflammatory mediators.

Changes in the serum levels of immunoglobulin IgA, IgG, IgM, positive acute phase proteins and complement activity, as well as the lymphocyte subpopulations and the neutrophil leucocyte-related chemiluminescence in the blood of patients with apical granuloma as related to endodontic and surgical treatment were investigated. Measurements were performed on admission, and 7 days and 3 months after the treatment. Elevated IgM concentration, positive acute-phase protein levels and spontaneous whole-blood chemiluminescence were noted at admission. However, a significant decrease in the serum level of each of the six investigated acute-phase proteins, and in the spontaneous chemiluminescence of blood was observed during the 3-month follow-up period. The significant increase in serum complement activity following therapy suggests that complement fixation might have occurred in these patients. A significant increase in the ratio of early sheep erythrocyte rosette-forming lymphocytes was also observed. The results of this study provide evidence for complete recovery after elimination of local inflammation by proper endodontic treatment and apicectomy in patients with apical granuloma.

Spontaneous rosette formation of carp peripheral leucocytes with mammalian red blood cells.

The spontaneous rosette formation of peripheral leucocytes of carp with red blood cells of various mammals was investigated. Spontaneous rosette formation was observed with SRBCs, HRBCs and BRBCs with the frequency of rosette formation being higher with SRBCs and HRBCs than with BRBCs. Variations in surface morphology, in cell size of the rosette forming cells and in rosette formations were observed under light and scanning electron microscope. However, by examining Giemsa-stained preparates, the RFCs were identified as lymphocytes. The spontaneous rosette formation occurred gradually depend-ing on the incubation time. The spontaneous rosette formation was not specific to SRBCs and was observed at a temperature range of 0°C to 37°C. In these respects the rosette formation of carp lymphocytes differed from the earlier reported rosette formation of mam-malian lymphocytes.

The role of surface charges in reproduction

The influence of some of the well known technical variables on the E-rosette technique was examined using sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (AET). With AET treatment, E-rosette formation becomes less dependent on time and temperature and on the presence of serum. The mechanical stability of the rosettes is enhanced, and the number of SRBC attached to each rosette-forming lymphocyte (RFC) is markedly increased, leading to a sharper distinction between RFC and non-RFC. Ultimately, significantly more E-receptor carrying lymphocytes become detectable. Evidence is provided that the specificity of the E-rosette technique is unchanged after AET treatment of SRBC, in spite of the enhanced binding. A simple and reliable method for E-RFC identification is documented.

Morphologic and functional characterization of human peripheral blood T cells expressing the T cell receptor γ/δ*

The morphologic and functional characteristics of cells freshly isolated from human peripheral blood and bearing a T cell receptor (TcR) gamma/delta were analyzed. Cell preparations highly enriched for TcR gamma/delta+ cells were obtained by treatment of E rosette-forming lymphocytes with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. These preparations consisted of 64-82% TcR gamma/delta+ lymphocytes, as indicated by the sum of cells reacting with the BB3 and A13 mAb which define two distinct, nonoverlapping, TcR gamma/delta+ cell subsets in the peripheral blood. TcR gamma/delta cells were able to form conjugates with the natural killer-sensitive K-562 and with the natural killer-resistant HL-60-R tumor cell lines. The cytochemical localization of lysosomal acid hydrolases showed that 95%-98% of the cells in the TcR gamma/delta+ preparations had the morphologic features of granular lymphocytes. Moreover, electron microscopy analyses showed that TcR gamma/delta+ cells had electron-dense granules dispersed in the cytoplasm and a variety of smooth vesicles, a morphology identical to that of other CD3- or CD3+ granular lymphocyte subsets. Freshly isolated TcR gamma/delta+ cells were unable to lyse K-562 and natural killer-resistant targets, such as HL-60-R and P815. However, low levels of target cell lysis were observed upon triggering of the effectors by anti-CD3 TcR mAb or by lectin. After short-term culture with interleukin 2, TcR gamma/delta+ cells acquired a strong cytolytic activity against K-562 and HL-60-R target cells in the absence of triggering stimuli, and also displayed high levels of cytolytic activity against P815 in the presence of anti-CD3/TcR mAb.

Theophylline-Resistant and Theophylline-Sensitive “Active” and “Total” E Rosette-Forming Lymphocytes in Patients with Systemic Scleroderma

Using the E rosette test and its modification with theophylline, we have studied T regulatory lymphocytes in various forms of systemic scleroderma. Mean percentages of active rosette-forming cells (ARFC) as well as the fraction resistant to theophylline incubation (ARFC-res) were significantly decreased, irrespective of the variety of the disease, compared to the age-matched controls. Late ("cold") rosette-forming fractions were unimpaired. The theophylline-sensitive fraction of total rosette-forming cells (TRFC-sens), which contains mainly cells from the suppressor circuit, was found to be lowered in all patients groups studied, whereas the ARFC-sens fraction was significantly decreased only in patients with diffuse scleroderma over 50 years of age, in whom there was a tendency to a more severe course, as manifested by pronounced systemic organ involvement. The lowered values of E rosette tests were found in a majority of SSc patients and were correlated with the appearance in the sera of factors capable of inhibiting ARFC formation by normal human peripheral blood T lymphocytes. Normal values of the E rosette test were related to the presence in the patients' sera of factors stimulating ARFC formation by normal lymphocytes. We surmise from the results that in SSc patients the T-cell defect is not only restricted to T suppressor cells but also refers to the active theophylline-resistant fraction containing mainly T inducer and T cytotoxic cells.

Further evidence for heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes: analysis using monoclonal antibodies to two distinct types of Fc gamma-receptor.

In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (Fc gamma Rs), one monospecific for IgG2 (Fc gamma 2R) and the other bispecific for IgG1 and IgG2 (Fc gamma 1/gamma 2R), when analyzed by EA-rosette assay. These Fc gamma Rs on the cells were further studied by using two monoclonal antibodies toward the Fc gamma Rs on guinea pig peritoneal macrophages (anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibody). The anti-Fc gamma 1/gamma 2R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fc gamma 2R but not by anti-Fc gamma 1/gamma 2R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fc gamma 2R and anti-Fc gamma 1/gamma 2R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-Fc gamma R antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of Fc gamma R, Fc gamma 1/gamma 2R, and Fc gamma 2R, are expressed on the cells. The determination of these Fc gamma Rs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fc gamma 1/gamma 2R alone and 32% of the cells expressed both the Fc gamma Rs. On the other hand, about 12% of T lymphocytes was found to express Fc gamma 2R alone and the cells expressing Fc gamma 1/gamma 2R were in the minority (3.8%). T lymphocytes expressing both the Fc gamma Rs were not detected. These results show that guinea pig B lymphocytes bear two types of Fc gamma Rs and are heterogeneous with regard to their Fc gamma Rs and that T lymphocytes express Fc gamma 2R mainly.

Enhancement of antibody responses by IgD-binding factors induced by anti-IgD treatment of spleen cells.

Treatment of normal BALB/c splenocytes with anti-mouse IgD antibodies at 0 degrees C followed by incubation of the cells at 37 degrees C resulted in the formation of soluble factors that selectively inhibit rosette formation of lymphocytes bearing Fc delta receptors with IgD-coated erythrocytes (i.e., IgD-binding factors). Treatment of the same cells with anti-IgM antibodies failed to induce the formation of the factors. Analysis of cellular mechanisms indicated that polymerized surface IgD on B cells, as well as surface IgD-anti-IgD complexes shed from the B cells, induced T cells bearing Fc delta receptors to form IgD-binding factors. The factors formed by the anti-IgD treatment of splenic lymphocytes are composed of molecular mass species of 70 and 34 kDa, as estimated by gel filtration. Both the 70- and 34-kDa IgD-binding factors enhanced IgM and IgG1 plaque-forming cell responses of sheep erythrocyte-primed mouse spleen cells to the antigen.

Inhibition of human T lymphocyte E rosette formation by neutrophils and hydrogen peroxide. Differential sensitivity between helper and suppressor T lymphocytes.

Co-incubation of human mononuclear cells with small numbers of autologous polymorphonuclear leukocytes resulted in a ratio-dependent inhibition of T lymphocyte rosette formation with sheep erythrocytes (SRBC). Inhibition by polymorphonuclear leukocytes was reversed with catalase, implicating H2O2 or associated products as the suppressive agent(s). Exposing T lymphocytes directly to micromolar concentrations of H2O2 also inhibited subsequent rosette formation. Inhibition was dose-dependent between 40 and 320 microM H2O2 and was not due to cytotoxicity at those H2O2 concentrations. Kinetic studies demonstrated that after exposing T lymphocytes to H2O2 a proportion of cells appeared to be relatively resistant in that they retained their ability to form E rosettes. T cell phenotype analysis of these cells showed that, in contrast to untreated T cells, H2O2-treated T rosette-forming cells were almost exclusively of the helper/inducer phenotype. Analysis with the monoclonal antibody 4B4 revealed that H2O2-resistant T rosette-forming cells contained significantly fewer 4B4-positive cells than predicted for the T helper population, indicating that H2O2-resistant T cells might be a subset of helper/inducer T lymphocytes. The interaction between H2O2 and T cells was rapid but did not lead to loss of Leu-5b binding sites. Preliminary functional analysis indicates that interleukin 2 production and phytohemagglutinin-induced proliferation by the relatively H2O2-resistant T cells is similar to untreated T cells. In contrast, H2O2-treated T cells which lost their ability to form E rosettes produced undetectable levels of interleukin 2 and proliferated poorly in response to phytohemagglutinin. Finally, mononuclear cells pretreated with H2O2 and subsequently stimulated with mitogens demonstrated a preferential expansion of the T helper population with concomitant loss of T suppressors. Our results show that, although neutrophil-released H2O2 inhibits effective interactions between SRBC and T lymphocyte sheep erythrocyte receptors, there appears to be a population of T helper cells which is relatively resistant to H2O2 both in terms of SRBC rosette formation and response to mitogens. These data suggest that at sites of inflammation phagocyte-released H2O2 could exert immunoregulatory effects on T cells by altering T cell subset survival and allowing the function of a particular lymphocyte population to predominate.

Dog erythrocyte rosette-forming lymphocyte: blockage by OKT11 monoclonal antibody.

Human peripheral blood mononuclear cells (PBM) were separated into sheep erythrocyte rosette-forming (Es+) and non Es+ cells by the Ficoll-Hypaque gradient sedimentation method. Thirty-eight percent of the Es+ cells formed rosettes with dog erythrocytes and were designated as Es+Ed+ cells. The remaining Es+ cells were designated as Es+Ed- cells. Only a few non Es+ cells formed rosettes with dog erythrocytes. Among Es+Ed+ cells, T4 antigen-positive cells were observed approximately 1.7 times as often as T8 antigen-positive cells, when measured by staining with OKT4 or OKT8 monoclonal antibody. Among Es+Ed- cells, however, T4 and T8 antigen-positive cells were observed in almost equal proportion. Preincubation of PBM with OKT11 monoclonal antibody, but not with OKT4 monoclonal antibody, inhibited the rosette formation with dog as well as sheep erythrocytes. These results indicated that Es+Ed+ cells were a subpopulation of T-cells in which a majority of the cells were T4 antigen-positive, and that the binding sites of dog erythrocytes on human T-cells was closely linked with that of sheep erythrocytes.

Immuno-, neuro-, and general toxicologic animal studies on a synthetic pyrethroid: Cypermethrin

The toxicologic properties of the synthetic pyrethroid insecticide cypermethrin have been studied in animal experiments. In the course of acute and subchronic experiments performed by the administration of 1 2 and 2 4 , 1 20 , and 1 10 proportions of the oral LD 50 value, no considerable changes were found in the general toxicologic tests. The compound known to affect the central nervous system, however, induced only a mild enhancement of the central excitation level shown by the EEG investigations in the doses applied. In the immunotoxicologic studies an early and dose-dependent suppression was induced as a result of humoral immune response of rabbits immunised by S. typhi following the administration of cypermethrin. The cell-mediated immune response was also decreased. In rats the immune response determined by anti-sheep erythrocyte and antiovalbumin titer as well as by autologous rosette formation of spleen lymphocytes was hindered. The earliest detectable group of symptoms indicating the effect of a mild cypermethrin exposure is the appearance of the positivity of the immunotoxicologic tests. On the basis of the experimental findings the sensitive immunotoxicologic tests have been regarded as of great importance in the series of toxicologic methods.

Decreased cellular immune response in wasted but not in stunted children

BCG immunization was given to 114 rural Mexican children who had not responded with a positive reaction to a previous tuberculin test. A second tuberculin test was given to these children 5 weeks after the immunization. Heights, weights and upper-arm circumferences were also measured at these two time points. The diameter of induration resulting from the tuberculin test was measured. The percent of E rosette-forming lymphocytes and the mean number of sheep red blood cells (SRBC) per lymphocyte were also determined in 27 of these children. The diameter of the induration, the percent of rosette-forming lymphocytes and the mean number of SRBC per lymphocyte were positively associated with measures of wasting, but not with measures of stunting. We conclude that cell-mediated immunity is impaired in wasted, even when mild to moderate, but not in stunted children.

IgD-binding factors from mouse T lymphocytes.

Incubation of normal mouse splenic lymphocytes with dimeric mouse IgD results in the formation of T-cell factors that selectively inhibit rosette formation of Fc delta receptor-positive lymphocytes with IgD-coated erythrocytes. The rosette-inhibiting factors bound to IgD-Sepharose and were recovered by elution at acid pH. The results indicate that the rosette-inhibiting factors are IgD-binding factors. Neither IgE-binding factors nor IgG-binding factors inhibited IgD rosettes. The IgD-binding factors induced by IgD consist of two species of molecular weight 78,000 and 37,000, respectively. The source of IgD-binding factors is Lyt-1+ T cells bearing Fc delta receptors. It appears that the binding of IgD to the receptors on the T cells induces the formation of IgD-binding factors.

Selective IgA deficiency in a case of systemic lupus erythematosus

A 41-year-old female with high grade fever and polyarthralgia was diagnosed as SLE from polyarthralgia, leukopenia, LE cell and high titer anti-nuclear antibody. The serum IgA level was as long as 9mg/dl and unchanged by the treatment with prednisolone, while the serum IgG and IgM were increased and responded to the treatment.The persentage of E rosette-forming lymphocytes in the peripheral blood was 79% and the persentage of surface-IgA bearing cells in non E rosette-forming cells was 20%.When PWM-induced IgA synthesis by lymphocytes from normal control and the patient were measured, the level of IgA was about a quarter of normal synthesis. Co-culture of T cells from normal control and B cells from the patient resulted in reduced synthesis of IgA. Co-culture of the patient T cells and normal B cells also resulted in reduced synthesis of IgA. Thus the pathogenesis of IgA deficiency in the patients may be due to both B cells and T cells dysfunction.

Placental proteins in the diagnosis and evaluation of the "elusive" early pregnancy.

For many many years much time and effort has been invested to identify, purify, and quantify markers of significant reproductive events. The search for such markers has been recently intensified with the widespread acceptance of IVF and ET as a treatment regimen for the management of certain causes of infertility. Now it is possible to monitor ovarian folliculogenesis and time of ovulation, successful fertilization, and subsequent nidation and the ensuing pregnancy. Following successful fertilization and zygote development, EPF, a protein complex which suppresses in vitro lymphocyte rosette formation can be detected within 24 hours. Shortly after implantation hCG can be measured in the maternal circulation. The developing trophoblastic tissue continues to synthesize and secrete into the maternal circulation many varied proteins including SP1, PAPP-A, and PP5. To date hCG is the mainstay of rapid and early pregnancy diagnosis and is an accepted biochemical marker of successful implantation and trophoblastic activity. Combination of hCG and EPF measurement offers the clinician new insight into his infertile patient for now it is possible to monitor successful fertilization and implantation as separate independent events. Such diagnostic assays should also provide new insight into early embryo mortality rate at the pre- and postimplantation stage and hence indicate the selection pressures acting on an embryo. It is the application of these assays which has led to the definition of subclinical abortions and to the description of biochemical pregnancies. The value of these proteins for the management of compromised early pregnancies has to be more fully examined. Although the differentiation between a normal and abnormal pregnancy is more readily ascertained by serial sampling and analysis of the time taken for circulating levels to double (3). Preliminary data suggest the potential of EPF and PAPP-A measurement in such clinical conditions is enormous. Our studies suggest that PAPP-A, at present, is the only biochemical marker which can predict pregnancy abortion, even in the presence of normal ultrasound scans, many weeks prior to the event, thus implying a vital biological role for PAPP-A in normal pregnancy (52). Despite the need for more intensive evaluation of these early pregnancy indicators the search for that "demmed elusive" marker has more than "Those Frenchies" earnestly looking for him here and there.

Human semen inhibits T rosette formation through an opiate mediated mechanism.

Seminal plasma contains high levels of opioid peptides and both seminal plasma and endogenous opioids can influence the immune system. In order to investigate whether these two findings can be related, semen was collected from 7 normal subjects, and assayed for beta-endorphin content and for its in vitro ability to inhibit the total T rosette formation of human lymphocytes in the presence or in the absence of 10(-6) M naloxone, an universal opiate antagonist. The results were as follows: 1) immunoreactive beta-endorphin content in seminal plasma was 4 to 12 times higher than the peripheral plasma levels detected in the same subjects (76.1 +/- 42.1 SD vs 10.5 +/- 2.0 SD pg/ml); 2) increasing concentrations of seminal plasma (1%, 5%, and 10%) in RPMI 1640 significantly depressed the T rosette formation ability of lymphocytes; and 3) the simultaneous addition to the incubation mixture of 10(-6) M naloxone prevented the phenomenon, while naloxone per se was ineffective. The possibility that endogenous opioids may play a role in the immunomodulatory action of human semen is suggested.

Effect of methionine-enkephalin plus ZnCl 2 on active T cell rosettes

Methionine-enkephalin (Met-Enk) and ZnCl 2 in combination enhances active T cell rosette formation of human peripheral blood lymphocytes to a greater degree than either of the agents used separately. Enhancement of rosette formation by Met-Enk plus ZnCl 2 was not inhibited by the opiate receptor antagonist naloxone but was completely blocked by the zinc chelator 1, 10-phenathroline. The results suggest a relationship between zinc and Met-Enk and that zinc may be a modulator of enkephalin binding and function in the immune system as well as the nervous system.

Lymphocyte bacterial rosette test: Methodological details and effect of metabolic inhibitors and divalent cations

Lymphocytes bind certain bacteria. This property has been utilized in the lymphocyte bacterial rosette assay to identify T and B cell subsets. Here we determined the optimum conditions for the assay, studied the effect of metabolic inhibitors and divalent cations and compared bacterial rosette-forming lymphocyte subpopulations with those defined with monoclonal antibodies. The strains used were Brucella melitensis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli. Optimum attachment was obtained at 4°C in 6% BSA with ultrasonicated bacteria. Pretreatment of lymphocytes with the microfilament-disruptive drug cytochalasin B suppressed the binding of bacteria, whereas colchicine (inhibitor of microtubules), puromycin (inhibitor of translation), sodium azide (inhibitor of oxidative phosphorylation) and 2-deoxyglucose (inhibitor of glycolysis) had no effect. Divalent cations were required for the attachment of bacteria. B. melitensis bound to DR-positive cells, whereas the other bacterial strains rosetted OKT4-, OKT8- and DR-positive cells without exhibiting helper or suppressor T cell or B lymphocyte specificity.

Analysis of a sheep anti-pig T lymphoblast serum with specificity for E rosette-forming lymphocytes

Sheep antibodies against a pig E-rosette-forming lymphoblastic T lymphoma raised by two intravenous injections of 10 10 cells showed little lymphocytotoxic activity which could be absorbed with red cells, alveolar macrophages or kidney or liver cell homogenates. Bone marrow absorption yielded subpopulation specific antibody which binds to E rosette-forming cells (E.RFC) using either complement-mediated cytotoxicity or indirect antiglobulin rosette formation. In 30 blood lymphocyte preparations from 20 pigs with a range of ∼20–85% E rosettes the mean E% 43.5 ± 2.7 agreed with the % antigen ∗+ cells by cytotoxicity mean = 42.6 ± 2.7 and in each individual sample these figures also agreed closely. In samples of blood lymphocytes enriched and depleted for E rosettes, results of %E ∗+ also agreed closely with % antigen ∗+ cells. This relationship also held for thymocytes and the specific antibodies could be completely absorbed with thymocytes. These data show that the antibody identified peripheral and thymic E.RFC. Bound to lymphocytes the antibody inhibited E rosette-formation with sheep red blood cells (SRBC) in saline (S) and dextran (DS) and with pig RBC in dextran and in Ficoll, but did not affect B cells shown by immunofluorescence, direct antiglobulin rosette formation or Fc rosette-formation, either in saline or dextran, (which include Tγ cells). E rosette inhibition was dependent on antibody concentration, showing single and double sigmoid curves for S and DS rosettes respectively, consistent with differing ease of inhibition of the strong and weak rosette formation. The same spectrum of inhibition of rosette formation by antibody binding followed subsequent incubation with C′ 6-deficient rabbit serum, but with C′-sufficient serum resulted in loss of cells which require dextran for Fc rosette-formation (Tγ). Thus the serum reveals E rosette-forming T cells and their subpopulations, perhaps by binding to the SRBC receptor.

Lymphocyte subpopulations in multiple sclerosis: Spontaneous and mitogen-induced activity

Lymphocytes of chronic progressive multiple sclerosis (MS) patients and healthy persons were isolated by nylon-wool column filtration followed by density centrifugation. Incubating part of these lymphocytes with 2-aminoethyliso thiouronium bromide hydrobromide-treated sheep erythrocytes resulted in two other subfractions, the rosette-forming (E +) lymphocytes and the nonrosette-forming (E −) lymphocytes. Increased spontaneous activity was observed with the nonfractionated lymphocyte populations and with the E +- and E −-cell fractions of MS patients compared to the corresponding cell populations of controls following a culture period of one or several days. Phytohemagglutinin- and pokeweed mitogen-induced stimulation of these different lymphocyte fractions was the same in MS patients and controls. No influence of disease activity could be found on the immune parameters. Characterization of the different lymphocyte populations with monoclonal anti-human T-cell antibodies demonstrated a prevalence for helper T cells in forming rosettes.

Thermostable erythrocyte rosette-forming lymphocytes in hereditary hemochromatosis. I. Identification in peripheral blood.

Although the immunoregulatory role of iron has been demonstrated in vitro, evidence for a similar role in vivo is controversial. We have, therefore, studied certain functional and structural properties of lymphocytes in hereditary (idiopathic) hemochromatosis (HH), a disease characterized by iron overload. T- and B-lymphocyte percentages in peripheral blood, serum immunoglobulin levels, and proliferative responses of peripheral blood mononuclear cells (PBM) to lectins were comparable with those of controls. Furthermore, HH serum with elevated iron concentrations did not significantly alter proliferative responses of normal lymphocytes to mitogens. In contrast to those normal findings was the identification of a subset of T lymphocytes in HH that formed rosettes with sheep red blood cells (SRC) at 37 degrees C in abnormally high numbers. Those lymphocytes that formed thermostable erythrocyte rosettes (TE-R) were not immature thymocytes, activated T lymphocytes, or an artifact of passive attachment of anti-SRC antibodies to the HH lymphocyte surface. Their presence did not correlate with a concentration of iron in the serum, the length of treatment, or the presence of the HLA antigen, A3. We conclude that the cellular expression of HH may be detected not as an immunological abnormality, but rather as an abnormality in receptor expression.