Bursa-independent origin of Fc IgG receptor-bearing lymphocytes in chickens.

On the basis of performed studies the following observations indicate that there is a bursa-independent origin of lymphocytes with Fc IgG receptors: (a) the first appearance of Fc positive cells is in fetal liver and bone marrow, (b) a relative low percentage of Fc receptor cells, as compared to B cells, occurs in the bursa during the embryonic and post-hatching period, (c) a far lower percentage of Fc positive cells than B cells is found in the spleen, (d) a normal level of Fc positive cells occurs in bursectomized birds, (e) a majority of these cells do not possess thymic or bursa antigen or surface immunoglobulin.     

Biological significance of Fc receptor-bearing cells among activated T lymphocytes.

Lethally irradiated mice injected with syngeneic thymocytes and immunized with protein antigens develop specific helper T cells. If injected with semiallogeneic thymocytes, such mice generate H-2 antigen-specific cytotoxic T cells. Most spleen cells from these chimeric mice possess Fc receptors. The present results demonstrate that the development of Fc-receptor-bearing cells in thymocyte-injected irradiation chimeras seemingly is due to the physiological conditions in the mice rather than to the specific immunization. As a corollary, both helper T cells and cytotoxic T cells did not have Fc receptors, at least not in their effector state. Thus, Fc receptors on T cells would seem irrelevant to their immune function.     

Vaccination with Mycobacterium bovis BCG affects the distribution of Fc receptor-bearing T lymphocytes in experimental pulmonary tuberculosis.

Inbred strain 2 guinea pigs were vaccinated with Mycobacterium bovis BCG or were left unvaccinated and challenged 6 weeks later by the respiratory route with virulent Mycobacterium tuberculosis. By using a double rosette assay with isotype-specific antibody-coated ox and uncoated rabbit erythrocytes, the proportions of T lymphocytes bearing Fc receptors for immunoglobulin G (IgG) (T gamma cells) or IgM (T mu cells) were quantified in tissues taken from animals that were killed within 4 weeks postchallenge. Tuberculin reactivity in vivo and in vitro and antimycobacterial resistance were also measured. BCG vaccination protected the guinea pigs and resulted in significantly enhanced proportions of T mu cells in the blood during the first 3 weeks and in the spleen during weeks 2 and 3 postchallenge. Levels of T gamma cells declined in all tissues during the first 3 weeks of infection and were unaffected by prior vaccination with BCG. Increased proportions of T mu cells in the blood were accompanied by dramatic tuberculin skin reactions and purified protein derivative-induced lymphoproliferation in BCG-vaccinated guinea pigs during the first 2 weeks following virulent pulmonary challenge. Peak levels of T mu cells in the spleens of vaccinated animals at 2 weeks coincided with the first appearance of virulent mycobacteria in that organ. BCG vaccination appears to influence immunoregulatory events in pulmonary tuberculosis through effects on the distribution of IgM Fc receptor-bearing (T mu cell) T lymphocytes.     

Different rosette assays for detecting Fc receptor-bearing lymphocytes measure different subpopulations

The capacity of purified lymphocytes from human peripheral blood to bind the Fc portion of IgG was investigated by the rosette technique using ox erythrocytes sensitized with rabbit anti-ox IgG (EA_ox) and human erythrocytes sensitized with anti-CD IgG (EA_CD). With unfractionated lymphocytes EA_ox always detected more rosette-forming cells (RFC) than did EA_CD; however, in lymphocyte populations specifically depleted of B lymphocytes by passage through copolymer styrene bead columns, the proportion of rosettes formed with EA_ox and with EA_CD was the same. Double labelling for Fc receptors and surface immunoglobulin (SIg) demonstrated that most of the lymphocytes which formed rosettes with either EA_ox or EA_CD also carried SIg. Pre-incubation of lymphocytes at 37° to remove heatlabile SIg did not affect their ability to form EA rosettes but reduced the proportion of SIg-bearing cells. Following pre-incubation a significant proportion of EA_ox RFC still remained SIg positive but the lymphocytes which formed rosettes with EA_CD no longer carried SIg.

These studies suggest that rosette formation with EA_CD detects only `K' lymphocytes (non-T, non-B cells bearing heat-labile SIg) while EA<sub>ox</sub> detects some B lymphocytes as well. By reducing the amount of IgG bound to ox erythrocytes sensitivity was reduced to the point where EA<sub>ox</sub> no longer formed rosettes with B lymphocytes but still detected `K' lymphocytes indicating either a qualitative or quantitative difference between the Fc receptors on B and `K' lymphocytes. Treatment of lymphocytes with trypsin decreased the percentage of rosettes formed with EA<sub>ox</sub> but not with EA<sub>CD</sub> supporting the conclusion that there is a structural difference between the Fc receptors on B and `K' lymphocytes although a difference in receptor density is not excluded.

When EA_CD and fluorescein-labelled ox erythrocytes sensitized with a low concentration of rabbit anti-ox IgG were mixed, most RFC bound both the indicator erythrocytes simultaneously suggesting that the Fc receptors on `K' lymphocytes do not exhibit species specificity.

     

Interaction of anti-staphylococcal protein A antisera with Fc receptor-bearing human normal lymphocytes.

Staphylococcal protein A (SpA) is known to bind the Fc region of IgG of most mammalians and to possess biologic activity both in vivo and in vitro, where it acts as a lymphocyte polyclonal mitogen. Its binding to the Fc gamma portion bears many features of the antibody-antigen interaction, such as the dissociation constant, lattice formation, and complement activation. Moreover, SpA seems to compete with membrane Fc receptors for IgG so that the possibility of an interaction with the same CH domain(s) of IgG can be considered. In the present study, evidence is given that anti-SpA antisera obtained from chickens and rabbits are able to inhibit EA rosette formation by normal human lymphocytes and that they are able to recognize, with immunofluorescent staining, a subpopulation of normal human peripheral blood lymphocytes (PBL) that closely resembles that of EA rosette-forming cells (RFC). Moreover, the depletion of EA RFC by means of a single gradient centrifugation is accomplished by the parallel depletion of PBL stainable by anti-SpA antisera. The relevance of these results in the hypothesis of a similarity between the combining sites of SpA and membrane Fc receptor(s) for IgG is discussed.     

A novel lymphokine derived from human IgG Fc receptor-bearing B cells: its suppressive effect on activated T and B lymphocytes including tumour cells in vitro.

Peripheral blood FcR gamma-bearing human B cells, but neither T cells nor adherent cells, produce an immunoregulatory lymphokine after receiving the stimulation of FcR gamma by immune complexes--antibody-sensitized erythrocytes (EA). This factor suppresses polyclonal immunoglobulin (Ig) production of B cells to pokeweed mitogen (PWM) and Nocardia opaca delipidated cell mitogen (NDCM), indicating that not only B cells, but also T cells are targets for this factor. It also inhibits the proliferation of mitogen-activated mononuclear and T and B tumour cells in vitro. The inhibitory effect on tumour cell growth is cytostatic, but not cytotoxic as in the case of lymphotoxin (LT). All these suppressive effects are observed in a HLA-non-restricted manner. Irradiation (2000 rads) of FcR gamma + B cells does not inhibit the production of this suppressive factor, implying that DNA synthesis is unnecessary. Nonstimulated FcR gamma + B cells retain the precursor activity for Ig-forming cells, since mononuclear cells untreated with EA respond to the mitogens, resulting in Ig production. However, it is worthy to note that they lose the activity when stimulated with immune complexes. Thus, the property obtained from human FcR gamma + B cells is similar to, but distinct from, a murine suppressive B-cell factor (SBF) prepared by the same procedure as for the human factor. Nevertheless, the observation in the present studies on the human analogue to murine SBF suggests that this factor, tentatively termed human SBF, appears to be a novel lymphokine which is different from any other factors, including LT, and that FcR-bearing B cells play an important role in the immunoregulatory mechanism in humans, as in the case of mice.     

Cell populations in leucocyte adherence inhibition: requirement for T lymphocytes with IgG Fc receptors.

The subset identity of T lymphocytes participating in the leucocyte adherence inhibition (LAI) reaction was investigated. Humans were immunized with keyhole limpet haemocyanin (KLH) and their cellular immunity was tested by means of the haemocytometer variant of the LAI method. Their lymphocytes were fractionated by rosetting methods employing neuraminidase-treated sheep erythrocytes and IgG- or IgM-coated ox erythrocytes. The T lymphocytes rosetted by IgG-coated ox cells (T gamma) reacted with KLH to give specific LAI reactions. Non-T gamma cells failed to react. The T gamma cells released a lymphokine which caused an LAI reaction of T lymphocytes from non-immunized donors. Immune non-T gamma cells, when incubated with KLH yielded inactive supernates. The normal cells which gave positive LAI responses to the lymphokine also proved to belong exclusively to the T gamma subclass. Cells positively selected with IgM-coated ox cells (T micro) were inactive while the non-T micro lymphocytes behaved like the T gamma cells. It was shown that the activity was confined to the T gamma subset throughout the time course of a primary immune response. Thus, LAI reactivity appears to be a property of a very small subclass of lymphocytes which communicate with each other by means of a soluble factor.     

OKT3-activated locomotion of human blood lymphocytes: a phenomenon requiring contact of T cells with Fc receptor-bearing cells.

The OKT3 antibody activates locomotion of human blood T lymphocytes as measured by polarization assays and invasion of collagen gels. The proportion of motile cells increases during a period of 24-48 hr of culture, even following only a brief initial contact with OKT3. The motile cells are the growing population. Locomotion activation is cell-density dependent. Studies with surface-bound mitogens, namely substratum-bound OKT3 and Con A-Sepharose, showed that only lymphocytes in direct contact with the mitogen acquired locomotor capacity. Those separated from it by a cell-impermeable filter were not activated. The response to OKT3 requires the whole antibody molecule. F(ab')2 fragments were inactive. Intact normal human IgG, but not its F(ab')2 fragments, blocked the response. Removal of RFc gamma + cells from the population by rosetting with IgG-Ab-coated sheep red cells prevented the response. These findings suggest that an RFc gamma + population has to be present for T cells to become activated to locomotion by OKT3, and that the OKT3 antibody links the T cell to an FcR+ cell, cell-to-cell contact being essential for activating the locomotor response.     

Lowered responsiveness of bronchoalveolar lavage T lymphocytes in hypersensitivity pneumonitis.

We previously reported that Trichosporon cutaneum was the major causative antigen of summer-type hypersensitivity pneumonitis (HP) in Japan. In summer-type HP patients, we noticed that the proliferative responses of bronchoalveolar lavage (BAL) lymphocytes to phytohemagglutinin (PHA) and concanavalin A were significantly lower than those of the peripheral blood lymphocytes of the same patients. It was shown in this study that the low response of BAL lymphocytes was due to an intrinsic lowering of the responsiveness of the T cells. Results of the mixed culture experiments, in which the responses to mitogens of BAL and peripheral blood T cells mixed with either alveolar macrophages or blood monocytes were compared, indicated that the decreased proliferative response was due neither to the suppressive effect nor to defects in accessory function of the alveolar macrophages. BAL T cells did not act as suppressor cells when they were added to the culture of peripheral T cells. The decreased proliferative response was not due to the dominance of CD8+ T cells frequently seen in BAL cells of HP patients, because both CD4+ and CD8+ T cells separated from BAL cells of HP patients showed lower responsiveness than those of peripheral blood T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

A major subpopulation of Fc receptor-bearing lymphocytes revealed by rosette formation in dextran media: studies with pig, sheep and rat lymphocytes

Blood lymphocytes from young pigs which formed 9.1 +/-0.7% Fc rosettes (mean +/- S.E., range 4.1-16.0), with rabbit and pig immunoglobulin-coated indicator cells used in optimum conditions in phosphate-buffered saline (PBS), formed 32.6 +/- 1.8% (16.9-44.3) in the presence of 4% dextran (DFc). The proportion of DFc+/Fc+ lymphocytes varied from 2.2 to 5.9 (3.8 +/- 0.3). Compared with the PBS test, in dextran rosettes are formed with more lymphocytes and with red cells coated with less immunoglobulin. Ficoll at 14% gave similar enhancement. Dextran enhancement of Fc rosettes was also observed with sheep PBL, but not with rat thoracic duct lymphocytes. The FC portion of IgG is responsible for both Fc and DFc rosettes. Thus most Fc and DFc rosettes, formed with serum antibody-coated bovine RBC, are revealed by IgG coated but not F(ab')2-coated BRBC, and show dose-dependent inhibition in the presence of free pig IgG (complete at 5-10 mg/nl) but not IgM (up to 2 mg/ml). Separation of lymphocytes on nylon wool suggests that DFc rosettes are formed by B cells and some T cells. Over half of the weak Fc rosette-forming lymphocytes (DFc-Fc) elute in the non-adherent fraction, which contains few B cells, and therefore are a subpopulation of T cells.     

Surface markers on human activated T lymphocytes. I. Fc receptors for IgG and IgM

Expression of Fc gamma and Fc mu receptors on human peripheral blood T lymphocytes of two subsets with high (E early rosette forming presumably in vivo activated cells, TEe) and low affinity of E receptors (E late rosette forming presumable resting cells, TEl) was investigated. Different distribution pattern of T gamma and T mu cells in the both examined T cell subsets was found. Thus TEe and TEl subsets have been partially enriched in T mu and T gamma cells, respectively. Furthermore, the results obtained in the PHA-stimulated system have shown that Fc mu receptors do not function as the markers of T cell activation. However, in opposition to this finding Fc gamma receptors may be the early activation markers but only of T cells originally bearing high-affinity E receptors.     

Receptors for IgA on human lymphocytes. II. Organ distribution and relationships with other Fc receptor-bearing populations

Lymphocyte populations expressing receptors for IgA (RFc alpha) in several human lymphoid tissues were numerically evaluated using a highly sensitive and specific indicator system. The percentages of RFc alpha-bearing lymphocytes were, in thymus, 9%; bone marrow, 14%; tonsil, 38%; cord blood, 34%; and adult blood, 55%. In adult and cord blood, RFc alpha were primarily associated with the T-lymphocyte populations as defined by their ability to bind sheep erythrocytes (SE). A large percentage of the peripheral T-lymphocyte population expressed both RFc alpha and receptors for IgM (RFcmu), whereas few T lymphocytes expressing receptors for IgG also displayed RFc alpha. Although a small number of peripheral non-T cells also expressed RFc alpha, they appeared to have fewer or less avid receptors than the RFc alpha-bearing T lymphocyte population. Both bone marrow and tonsil lymphocytes of both T and non-T populations expressed RFc alpha. Furthermore, the RFc alpha-bearing non-T population in both tonsil and bone marrow was quite large, 39 and 26%, respectively. These studies document the ubiquity of RFc alpha and its expression on the same peripheral lymphocyte population as RFcmu and indicate that RFc alpha is associated with several functionally different lymphocyte subpopulations including those involved in regulation (help) and antibody-dependent cell cytotoxicity.     

Which Fc receptor-bearing cells are detected with the Ripley assay?

Mononuclear cells were isolated from peripheral blood and analysed with T and B markers (E rosettes and SIg) and on contaminating monocytes and PMN. The suspensions contained 63.3 +/- 4.8% T lymphocytes, 11.4 +/- 3.8% B lymphocytes, 9.0 +/- 5.5% null lymphocytes, 15.1 +/- 3.8% monocytes and 1.1 +/- 0.6% PMN. Of all cells, 27.6 +/- 12.1% formed EA rosettes with OR1R2 red cells coated with anti-CD Ripley. In preparations fixed after cytocentrifugation, the EA rosette-forming cells were studied with regard to esterase activity. The proportion of cells with detectable Fc receptors was further studied in purified T lymphocyte and in monocyte suspensions. Finally, EA rosettes were isolated by gradient centrifugation and the rosette forming cells studied with conventional T and B markers. All procedures gave corresponding results: on average 11-14% of the T lymphocytes and nearly 100% of the null cells formed EA rosettes, while only 2% of the B lymphocytes had detectable Fc receptors. Of the purified monocyte and PMN populations, on average 72 +/- 10.5 and 14.5 +/- 5.4%, respectively, formed EA rosettes. Thus, the Ripley assay, representing an important marker for null lymphocytes, cannot be regarded as specific for this population of white blood cells.     

Kinetics of accumulation of gamma delta receptor-bearing T lymphocytes in mice infected with live mycobacteria.

The kinetics of accumulation of T cells bearing the gamma delta heterodimer form of the T-cell receptor in mice infected with live Mycobacterium bovis BCG or M. tuberculosis was studied. Substantial numbers of gamma delta T cells accumulated in mice given primary mycobacterial infections, although this accumulation was in parallel to, but not preferential to, that of alpha beta receptor-bearing T cells. In contrast, no accumulation of gamma delta cells was observed in memory immune mice upon rechallenge, thus suggesting that gamma delta cells play no role in the anamnestic response. The results of the study show, further, that large accumulations of gamma delta T cells can also be induced by inoculation with oil adjuvant vehicles containing heat-killed mycobacteria, although not by inoculation of the heat-killed bacteria alone.     

Immune complexes and autoantibodies in silicosis

Serum specimens from 53 patients with silicosis were examined for the presence of antinuclear antibodies (ANA), rheumatoid factor (RF), immunoglobulins, and immune complexes. These humoral immunologic parameters were compared with radiographic changes and pulmonary function studies. A significant percentage of patients had an increased prevalence of ANA, RF, and immunoglobulin elevation (IgG, IgA). Immune complexes determined by the Raji-cell assay were detected in 31% of the patients. However, there was no significant correlation between any humoral immunologic abnormality and radiographic changes or declines in pulmonary function tests. These findings suggest that humoral immunologic abnormalities are not directly responsible for the lung changes in silicosis and cannot be used as “guides” to predict severity or progression of disease.     

Cellular immunity in pregnancy: subpopulations of T lymphocytes bearing Fc receptors for IgG and IgM in pregnant women.

Studies on the change of peripheral T and B lymphocytes and T cells bearing Fc receptors for IgG and IgM in pregnant women were performed by using rosette-formation tests. There was no significant difference in the proportion of T and B lymphocytes between pregnant and non-pregnant women. The percentage of T cells bearing Fc receptors for IgG in the T lymphocytes which are considered to have suppressive activity increased in the various stages of pregnancy and post-partum as compared with that in non-pregnant women. On the contrary, the percentage of T cells bearing Fc receptors for IgM in the T lymphocytes which have a helper function decreased in pregnant and post-partum women. The results of this investigation suggest that the depression of cell-mediated immunity during pregnancy depends on the qualitative change of T lymphocytes, i.e. increased suppressor and decreased helper T lymphocyte subpopulations.     

T lymphocytes bearing receptors for Fc of IgG in guinea pig peritoneal fluid. A model for human extrahaematic fluids.

Surface markers of lymphocytes from peritoneal fluid of the guinea pig are studied and compared with those of lymphocytes from thymus, peripheral blood and spleen. The great majority of peritoneal lymphocytes form rosettes with rabbit red cells and bear receptors for Fc of IgG (T Fc+ cells). Lymphocytes with these characteristics are poorly represented in blood, thymus and spleen. Guinea pig peritoneal lymphocytes can be compared, as far as surface markers are concerned, with lymphocytes from human cerebrospinal fluid and transudates.     

Serum cytokine profile in hepatitis C virus carriers presenting cryoglobulinaemia and non-organ-specific autoantibodies

This work investigated the serum cytokine profile (IL-2, IL-4, IL-5, IL-10, IFN-γ and BAFF) of hepatitis C virus (HCV) carriers with autoimmunity. Forty-seven HCV carriers and 28 healthy controls were evaluated. Cytokine levels were measured by ELISA. Patients and controls presented similar levels of IL-2, IL-4, IL-5, IL-10, IFN-γ and BAFF (p > 0.05). Cryoglobulinaemic HCV carriers had increased IL-2 (p = 0.013), IL-5 (p = 0.018) and BAFF (p = 0.050). IFN-γ level was decreased in HCV carriers with rheumatoid factor in comparison with those that were RF-seronegative (p = 0.035). Patients with β2GPI IgA antibodies when were compared with those without this autoantibody, had more serum IL-2 (p = 0.009), IL-5 (p = 0.018) and BAFF (p = 0.039). Interleukin-2 was increased in HCV carriers with positive ANA when they were compared with ANA-seronegative carriers (p = 0.044). Interleukins IL-4 and IL-10 were not associated with autoimmunity (P > 0.05). In HCV carriers, IL-2 was correlated with IL-5 (p < 0.0001) and IFN-γ (p = 0.015), and IL-5 with IFN-γ (p = 0.015). We concluded that the serum profile of cytokines in HCV carriers presenting autoimmune markers may be mainly represented by increased IL-2, IL-5 and BAFF.