Stimulation of expression of IgM and IgG receptors on human thymus lymphocytes after treatment with lactoferrin in vitro

It was established by immunofluorescence that lactoferrin, one of the heteroorganic thymus antigens, can stimulate the expression of Fc mu and Fcj receptors on thymus lymphocytes. The stimulating effect of lactoferrin on T mu cells is more pronounced with the level of these cells in the thymus being low. Its effect on Tj cells seems independent of their level in the thymus and may be related to their precursor differentiation. It can be assumed that one of the functions of lactoferrin in the thymus is to influence the process of differentiation of T mu and Tj cells and to regulate their level in the thymus. Lactoferrin, like other heteroorganic thymus antigens, may take part in the functional maturation of different subpopulations of thymocytes, including T mu and Tj thymus cells.     

Radioimmunoassay of IgG and IgM rheumatoid factors reacting with human IgG.

Although IgG rheumatoid factor may play a central role in the pathogenesis of rheumatoid arthritis, previously there have been no precise methods for its specific measurement in serum and synovial fluid. This paper describes a solid phase radioimmunoassay for the independent quantification of IgM and IgG rheumatoid factor reacting with the Fc fragment of human IgG. As measured by this assay, serum IgG rheumatoid factor levels differed significantly between patients with seropositive and seronegative rheumatoid arthritis and normal control subjects. In addition, several sera and joint fluids from patients with seropositive rheumatoid arthritis, even without vasculitis, were shown by gel chromatography to have acid-dissociable complexes of IgG rheumatoid factor suggestive of IgG-IgG dimer or trimer formation.     

Subpopulations of human T lymphocytes. XII. In vitro effect of agents modifying intracellular levels of cyclic nucleotides on T cells with receptors for IgM (T mu), IgG (T gamma), or IgA (T alpha).

Purified peripheral blood T lymphocytes were incubated with inducers of cyclic nucleotides and examined for the numbers of T cells with receptors for IgM (T mu), IgG (T gamma), or IgA (T alpha). Isoproterenol and theophylline, agents known to increase cAMP levels at 10(-3) to 10(-6) M concentrations, significantly decreased the number of T mu cells but had no effect on T gamma or T alpha cell numbers. This effect of isoproterenol could be completely blocked by a beta-adrenergic blocking agent, propranolol. Receptors for IgM on T mu cells regenerated after treatment with theophylline when cells were washed and further incubated at 37 degrees C over a period of 12 to 24 hr in the absence of theophylline. Phenylephrine, at 10(-3) to 10(-6) M concentrations, significantly increased the numbers of T mu cells but had no effect on T gamma or T alpha cell numbers. The effect of phenylephrine could be completely blocked with an alpha-adrenergic blocking agent, phentolamine. The significance of the results are discussed.     

Insoluble immune complexes suppress mitogen-induced proliferation of human T lymphocytes bearing Fc gamma receptors.

Human peripheral blood lymphocytes were mixed with erythrocyte-antibody (EA) complexes and separated into EA-rosette forming cell (EA-RFC)-enriched and EA-RFC-depleted suspensions. Thymidine incorporation of EA-RFC-enriched population in the presence of T cell mitogens (PHA, Con A, PWM) was about half of that of EA-RFC-depleted or of unseparated cells. The dose-response curves and kinetics of proliferation were found to be very similar in the three populations. Proliferative response of EA-RFC-enriched lymphocytes was strictly T cell dependent, although non-T cells were later recruited to incorporate thymidine. The interaction of T lymphocytes bearing surface receptors for IgG (T_G) with insoluble complexes followed by a post-binding temperature sensitive event, resulted in the modulation of Fc receptors associated with an impaired proliferative response to PHA, Con A, and PWM, without significant change in metabolic cell activity as shown by cell viability, sponaneous leucine incorporation, or β₂ microglobulin release.     

Receptors for IgM on certain human B lymphocytes.

A receptor for IgM was demonstrated on the surface of human B lymphocytes by using a rosette technique with ox erythrocytes coated with rabbit IgM antibody (EAM). Lymphocytes forming rosettes with EAM did not bind sheep red cells, had membrane Ia-like antigens and, in some instances, surface immunoglobulin. The specificity of EAM rosettes was confirmed by inhibition experiments with purified human Ig. IgM but not IgG molecules inhibited the rosette reaction. In addition, inhibition of EAM rosettes with IgM fragments showed that the receptor has affinity for a part of the molecule located in the Fc portion. By analogy with the receptors previously found on certain human T cells, receptors for IgM were not detected on freshly isolated B cells, but were expressed after overnight culture in IgM-free media. Studies on different human lymphoid tissues showed that IgM receptors are expressed on a limited percentage of both circulating and noncirculating B cells. In addition to normal B cells, the malignant B cells of a majority of cases of chronic lymphocytic leukemia expressed the receptors for IgM.     

Guinea pig peritoneal macrophages. Differential effects of lectins on interaction with IgG immunoglobulins

Guinea pig peritoneal macrophages have on their surface two receptors, one (Fcγ₁/γ₂R) binding both guinea pig IgG1 and IgG2 and the second (Fcγ₂R) binding only IgG2 immunoglobulins. We have previously shown that treatment of macrophages with neuraminidase or glycosylation inhibitors affects, in a different way, the binding of guinea pig IgG1, IgG2, and rabbit IgG. In the present study we have shown that pretreatment of guinea pig macrophages with lectins (Con A, WGA, and PNA) also has a different effect on the interaction of the cells with IgG. The lectins increased the binding of guinea pig IgG1, whereas rabbit IgG and guinea pig IgG2 were bound with a lower efficiency than in the case of control cells. Since sialic acid residues seem to modulate the activity of receptors and WGA interacts with sialylated oligosaccharides, we determined the IgG-binding characteristics for WGA-pretreated macrophages. We found that the increase in IgG1-binding ability was caused by an increase in the value of K_app, but the number of IgG-binding sites was lower than in the control cells. In the case of rabbit IgG and guinea pig IgG2 we observed a decrease of both the value of K_app and the number of IgG-binding sites. WGA did not interact directly with the Fcγ receptor. The results of our former papers and the different effects of lectins of various specificities described in this paper suggest different positions of Fcγ₁/γ₂ and Fcγ₂R in the plane of the plane of the macrophage membrane in respect to various membrane glycoconjugates. Interaction of IgG with macrophage Fcγ receptors depends in a different way on glycoconjugates on the surface of the macrophage. Our results suggest that changes in glycosylation of macrophage surface glycoconjugates may be used by the cell for regulating the binding activities of the macrophage Fcγ receptors.     

Studies of surface immunoglobulins on human B lymphocytes. I. Dissociation of cell-bound immunoglobulins with acid pH or at 37 degrees C.

Lymphocyte preparations isolated from the human peripheral blood were exposed to different acid pH or incubated at 37 degrees C and the presence of immunoglobulin (Ig) on the cell surface was examined by immunofluorescence (IF) tests. Subsequently, such treated cells were incubated in the autologous serum or in the purified IgG, IgA or IgM proteins and their ability to bind each class of Ig was examined. The results showed that IgG molecules dissociated from large proportions of IgG-positive cells upon exposure to pH 4 at 1 degrees C for 1 min or upon incubation at 37 degrees C for 20 min. The cells from which IgG had been dissociated could again combine with IgG, whereupon the number of positive cells increased, being restored to the number of equivalent to or higher than those before acid or 37 degrees C treatment. These results indicated that the treatment could elute the cell-bound IgG present on the cell and that the receptor sites were not degraded by the treatment and could combine with IgG. These cell-bound IgG were observed not only on the monocytes, but also on the small lymphocytes. It was also found that certain proportions of mononuclear cells carried the cell-bound IgA that could be dissociated with acid pH or 37 degrees C. No cell-bound IgM was observed on any mononuclear cells. Microscopic observations before and after acid or 37 degrees C treatment revealed that the staining distribution of the cell-bound IgG and IgA on the cell was granular, appearing as a discontinuous fluorescence ring and forming multiple aggregates but no typical polar caps on warming. In contrast, IgG, IgA, and IgM stable to acid or 37 degrees C treatment were found on the lymphocytes but not on the monocytes, and their staining distribution was uniformaly diffuse, appearing as a continuous ring and forming a typical cap on warming. Exposure of the cells to pH 4 or 37 degrees C could also elute the cell-bound IgG passively adsorbed to the human lymphoid cells in a culture, but did not affect the intrinsic S.Ig on the lymphoid cells in a culture or on the lymphoma cells. These results indicate that the exposure of the cells to acid pH or to 37 degrees C may enable us to detect unfailingly S.Ig lymphocytes by removing the cell-bound IgG and IgA present on the monocytes and/or lymphocytes. Thus, an average value of approximately 10% was obtained for the S.Ig lymphocyte in the lymphocyte preparations from 11 healthy individuals. In addition, the results provided the evidence that, even in normal peripheral blood lymphocytes, there may be a population of B lymphocytes which lack the S.Ig but carry the cell-bound Ig.     

Survival of human T and B lymphocytes after X-irradiation.

The survival of unstimulated human T and B lymphocytes after X-irradiation in vitro was measured by Trypan Blue dye exclusion over a period of four days. B cell numbers were observed to decline rapidly even after relatively low doses, but T cell numbers fell much more slowly. A comparison of the percentage survival 96 hours after irradiation shows that in this system T cells are between approximately 2 and 5 times more resistant than B cells. Data for interphase death after 48 hours are compared with cytogenetic data for interphase loss of PHA-stimulated human lymphocytes and are shown to be in broad agreement at radiation doses below 400 rad. It is suggested that at higher doses mitotic delay may be increasingly important leading to selection of non-irradiated cells at 48 hours.     

CD16 on human gamma delta 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 gamma delta T cells. CD16 is expressed by the majority of gamma delta T cells in peripheral blood and by part of the gamma delta T cell clones. The amount of CD16 expressed on gamma delta T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated gamma delta T cells. Like CD16 on CD3- CD16+ natural killer (NK) cells, CD16 on gamma delta T cells can act as an activation site triggering cytotoxic activity. CD16+ gamma delta T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of gamma delta 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 Fc gamma R+ target cells by CD16+ gamma delta T cell clones. The mouse IgG-isotype specificity of CD16 on gamma delta 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 gamma delta T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.     

Immunophenotype of human lymphocytes after interaction with mesenchymal stromal cells

Human multipotent mesenchymal stromal cells (MMSCs) were cocultured with allogenic blood-born mononuclear cells (MNCs). The MNCs consisted of cells that differed in their maturity or functional state, such as lymphocytes from adult peripheral blood vs. umbilical cord blood (cb) or nonstimulated vs. phytohemagglutinin (PHA)-activated lymphocytes from peripheral blood, respectively. The share of T, B, and natural killer (NK) cells or T cell subsets within the initial MNCs or cbMNCs were within physiological reference range for adult peripheral blood. After coculturing with the MMSCs, the populations of B cells decreased in both MNCs and cbMNCs, whereas the populations of the T and NK cells decreased among cbMNC only (p < 0.05). A decrease in the subset of T-NK cells was observed in the T cells of both MNCs and cbMNCs. In the coculture of MMSCs and PHA-MNCs, we found decrease in the number of CD8⁺ and HLA-DR⁺ cells and an increase in the number of CD25⁺ lymphocytes compared to monocultured PHA-MNCs. Our data show that the interaction with MMSCs did not substantially modify the composition of allogenic lymphocytes independent of their maturation (MNCs vs. cbMNCs) or activation (MNCs vs. PHA-MNCs), and the means were within the physiological limits. Moreover, exposure to the MMSCs did not reduce the viability of lymphocytes and even promoted the survival of cells in case of cbMNCs.     

Separate Fc-receptors for immunoglogulins IgG2a and IgG2b on an established cell line of mouse macrophages.

The specificity of Fe-receptors on IC-21 cells, an established line of mouse peritoneal macrophages with antibody-dependent effector cell activity has been examined. Only IgG2a and IgG2b myeloma proteins bound readily to IC-21 Fc-receptors, the former in nonaggregated as well as aggregated form, the latter only as aggregated complexes. Thus, IgG2a bound in a manner characteristic of classically defined cytophilic antibody, whereas the binding of IgG2b appeared to be mediated by Fc-receptors for antigen-antibody complexes. Evidence is presented in support of the view that IC-21 macrophages possess separate and distinct Fc-receptor sites for these two immunoglobulins.     

Selective modulation of two human monocyte Fc receptors for IgG by immobilized immune complexes

Two types of IgG FcR, FcRI and FcRII, are constitutively expressed by human monocytes. FcRI (identified by mAb 32.2) binds human (h) IgG, FcRII (identified by mAb IV.3) has a low affinity for hIgG but interacts strongly with murine (m) IgG1. These receptors can be assayed by using indicator E sensitized by hIgG (EA-hIgG) or mIgG1 (EA-mIgG1), respectively. We further characterized these two FcR by modulation studies by using substrate-immobilized immune complexes containing rabbit IgG, goat IgG, or one of the mouse Ig classes or subclasses. After incubating monocytes in microtiter wells containing such immune complexes, binding of the two types of indicator red cells on the apical surface of the monocytes was quantitated using a photometric assay employing the pseudoperoxidase activity of E. No effect on the binding of sensitized E was observed after incubation of monocytes with immune complexes containing mouse IgE, IgA, or IgM, or F(ab')2 fragments of rabbit IgG. High concentrations of immune complexes containing IgG of mouse, rabbit, or goat, however, were able to induce a decrease in binding of both types of sensitized E, suggestive of modulation of both FcRI and FcRII. At lower concentrations of immune complexes, more selective patterns of modulation emerged. Under these conditions, immune complexes containing mIgG1 or mIgG2b, or, surprisingly, goat IgG induced a selective decrease in the binding of EA-mIgG1 (FcRII modulation), while immune complexes containing mIgG2a or rabbit IgG mainly affected the binding of EA-hIgG (FcRI modulation). By using anti-FcR mAb IV.3, it was confirmed that FcRII was modulated from the apical surface of monocytes after incubation on immune complex coated substrates. Selectivity of FcR-modulation was demonstrated by showing that under these conditions binding of anti-C receptor mAb, and several other anti-monocyte mAb did not decrease.     

A receptor for IgA on human T lymphocytes.

Receptors for IgA antibody-antigen complexes were demonstrated on 2 to 18% (mean 6.7%) of human peripheral blood T cells. The proportion of cells bearing detectable IgA receptors was low in freshly prepared T cells and increased in number after 18 to 24 hr of culture similar to the time course of appearance of the Tmu receptor. These T receptors were shown to be distinctly different from Fc-IgM and Fc-IgG receptors on T cells by blocking studies with purified IgA, IgG, and IgM.