Nobuhiro Noro

Transglutaminase activity during the differentiation of macrophages.

Summary Transglutaminase (R-glutaminyl-peptide:amine γ-glutamyltransferase, E.C. 2,3,2,13) activity during the differentiation of murine leukemia cell lines (M1 cells) was investigated. Ml cells contained a significant transglutaminase activity of the tissue type. During the course of differentiation into mature macrophage-like M1 + cells induced with a protein inducer, the enzymatic activity was stimulated more than ten times as much as in the original undifferentiated Ml − cells. A remarkable enhancement of enzymatic activity was also observed when lipopolysaccharide was utilized as an inducer of differentiation. The enzymatic activity of undifferentiated M1 − cells was eluted at the region of M.W. ca. 80,000 as a single and symmetrical peak on Sepharose 4B column chromatography. By contrast, most of the activity in differentiated Ml + cells was eluted at the void volume under the same condition, though some activities were eluted at the same region as in Ml − cells. These data suggest that most of transglutaminase activity exists in the form of a high-molecular-weight complex with some cellular components in differentiated cells. The possible physiological significance of the enzyme in macrophage functions was discussed.

T and B lymphocytes with immunoglobulin E Fc receptors (FcϵR) in patients with nonallergic hyperimmunoglobulinemia E: Demonstration using a monoclonal antibody against FcϵR-associated antigen

Abstract T and B cells bearing Fc receptors for IgE (FcϵR) were studied in 7 patients with hyperimmunoglobulinemia E (2 with hyper IgE syndrome and 5 with Kimuras disease). FcϵR was detected by both rosette formation with IgE-coated red cells (Eo′-IgE) and immunofluorescence assay using H107 monoclonal antibody recognizing a determinant(s) associated with lymphocyte FcϵR. A high correlation was observed between the proportions of Eo′-IgE rosette-forming cells (RFC) and H107+ cells. All patients had a large number of FcϵR-positive cells (mean ± 1 SD; 9.7 ± 3.7% Eo′-IgE-RFC, 8.4 ± 3.4% H107+ cells) in contrast to those of 6 normal subjects (0.7 ± 1.2% Eo′-IgE-RFC, 0.3 ± 0.4% H107+ cells). In one patient with Kimuras disease, the presence of FcϵR-bearing T cells was confirmed by two-dimensional flow cytometry, using fluorescein isothiocyanate (FITC)-H107 and phycoerythrin (PE)-Leu-1. H107 antigens seemed to be expressed on both helper/inducer and suppressor T-cell populations. The direct analysis of FcϵR+ T cells by 2-D flow cytometry with H107 antibody may facilitate the study of hyperimmunoglobulinemia E.

Antibodies against viral proteins can be produced effectively in response to the increased uptake of alpha2-macroglobulin: Viral protein conjugate by macrophages☆

We have previously shown that foreign antigens conjugated to alpha 2-macroglobulin (alpha 2M) were effectively taken up by macrophages, which was followed by a remarkable increase in the proliferation of immune T cells. We present in this report that the production of antibodies against viral proteins can also be effectively achieved when viral proteins were conjugated to alpha 2M and fed to the in vitro immune system. Proteins derived from Kirstein murine sarcoma virus (Ki-MSV) were partially purified by gel chromatography and conjugated to alpha 2M by the action of proteinases. Viral proteins conjugated to alpha 2M were taken up by thioglycolate-induced murine peritoneal exudate cells (PEC) more effectively than the free viral proteins. Murine spleen cells were then added to PECs fed with free viral proteins or with alpha 2M: viral protein conjugates, and the cell mixtures were incubated for five days. Each culture medium was then assayed for a specific antibody production by enzyme-linked immunosorbent assay (ELISA). Figures based on such assays revealed that the production of antibodies against viral proteins was ten times higher when the proteins were fed to macrophages in conjugated forms with alpha 2M.

Murine T cell proliferation can be specifically augmented by macrophages fed with specific antigen: α-2-macroglobulin conjugate

Foreign antigens conjugated to alpha-2-Macroglobulin (alpha-2-M) were effectively taken up by murine macrophages via alpha-2-M receptors. Such effective internalization of alpha-2-M:antigen conjugate by macrophages resulted in a remarkable increase in its ability to activate murine immune T cells under the following conditions. After macrophages were incubated with alpha-2-M:antigen conjugate or unconjugated antigen, they were cultured with immune T cells and antigen-stimulated tritiated thymidine incorporation by T cells was measured. The stimulation of T cell proliferative response by macrophages fed with the conjugate was sixteen times higher than what was observed with macrophages pretreated in the same concentration of unconjugated antigen. These findings suggest a physiological function of alpha-2-M and give us a new technique of immunization.

IgA Binding Factors and Fc Receptors for IgA: Comparative Studies Between IgA and IgE Fc Receptor Systems

The expression of Fc receptors (FcR) for IgA (Fc alpha R) as well as for IgE (Fc epsilon R) on T lymphocytes (T cells) is enhanced or up regulated by the corresponding class of immunoglobulins (Ig). The production of class-specific regulatory factors binding to IgA and IgE (IgA binding factor [IgA-BF]; IgE binding factor [IgE-BF]) is also induced by these respective ligands. Murine IgA-BFs produced by a T hybridoma T2D4 and concanavalin A-activated spleen cells suppressed the in vitro IgA antibody responses of pokeweed mitogen-stimulated mouse spleen cells class-specifically. Human IgA antibody response was also suppressed by the murine IgA-BF. Similar suppressive IgA-BF is also produced by a human natural killer (NK)-like cell line (YT), which has no rearrangement of the T cell receptor beta-chain gene, indicating that non-T non-B/LGL cells may also be involved in the regulation of the class-specific antibody responses. It appears that, in human as well as murine systems, T- and NK-cells have the capacity to co-express multiple class-specific FcRs and to produce the corresponding immunoglobulin binding factors. While the Fc epsilon R expression is abnormally enhanced in the diseases with hyperimmunoglobulinemia E, disregulation of Fc alpha R is associated with certain human diseases involving the altered IgA regulation. In IgA nephropathy, which is characterized by increased serum IgA level and IgA deposition in the mesangium, there is an enhancement of the expression of Fc alpha R. In contrast, IgA failed to induce Fc alpha R significantly on the lymphocytes from the patients with selective IgA deficiency, indicating that Fc alpha R plays an important role in the IgA regulation in vivo.

Possible Involvement of Transglutaminase in Endocytosis and Antigen Presentation

Experiments were carried out to determine as to whether or not internalization of antigen is necessary for subsequent antigen presentation by accessory cells using monoamines which are known as transglutaminase (TGase) inhibitors. It was found that endoeytosis for immune complexes via Fc receptors such as sheep erythrocytes coated with IgG class antibody (EA) was different from receptor‐independent endoeytosis for soluble protein such as horse radish peroxidase (HRP) in the sensitivity to monoamines; methylamine inhibited the receptor‐dependent endoeytosis of immune complexes at a concentration of over 20 mM and the receptor‐independent endoeytosis of HRP at 2 mM, while dansylcadaverine (DC) inhibited both at a concentration of 100 μM. It was noteworthy that antigen‐specific T cell proliferation to splenic adherent cells pulsed with DNP9,6‐ovalbumin (DNP9,6‐OVA) was blocked strongly by DC as well, but weakly by methylamine. These results suggest the possibility that antigen presentation requires internalization of antigen by a mechanism such as receptor‐dependent endoeytosis for the subsequent reexpression of antigen on membranes. Furthermore, it was confirmed that TGase activity is high in peritoneal exeudate and spleen adherent cells, both of which have accessory cell activities for lymphocytes, suggesting the possibility that TGase might be involved intimately in receptor‐dependent endoeytosis and subsequent antigen presentation.

Synergistic interaction of a cytokine produced by embryonic fibroblasts and a lymphokine contained in Con A-stimulated spleen cell culture for murine macrophage differentiation: A model experiment using cell line cells, M1

The modulating activity of the culture supernatant of Con A-stimulated murine spleen cells for macrophages was investigated by using M-1 cells, which could differentiate into macrophage-like cells (referred to as M+1 hereafter), cocultured in a conditioned medium (CM) containing a differentiation factor (DF) obtained from the secondary culture of murine embryonic fibroblasts. DF induced Ia antigens on M-1 cells at a high rate in parallel with the appearance of Fc-receptor (FcR)-dependent phagocytic activity for erythrocytes coated with an antibody (EA). In contrast, Con A-sup alone had no modulating effect on M-1 cells. However, the Con A-sup stimulates synergistically M-1 cells with DF. Thus, coculture of M-1 cells with Con A-sup and DF generates M++1 cells which possess higher phagocytic activity than M+1 cells. These cells also exhibited stronger accessory cell activity than M+1 cells when tested for their promoting effect on IL-2 production by Sephadex G-10-passed spleen cells. The accessory cell activity of M++1 cells was further enhanced by culture with lymphocytes in the presence of indomethacin while that of M+1 cells did not change. These findings suggest that M-1 cells probably acquire potentiating, as well as inhibitory activity at the same time when cultured with DF and Con A-sup. The functional maturation caused by Con A-sup seemed to be associated with the expression of a receptor for a lymphokine, termed phagocytosis-augmenting factor (PAF) which is present in the Con A-sup. Such a receptor appeared to be common to macrophage lineage, since PAF in Con A-sup was absorbed out with splenic adherent cells and peritoneal exudate cells (PEC) in addition to M+1 cells, but not with nonadherent splenic lymphocytes or lymphoid cell line cells, such as EL-4 and L-1210. This fact suggests that PAF is different from interferon-gamma (IFN) which is known to modulate the function of lymphocytes. Inability of PAF to bind Cibacron Blue-Sepharose, unlike IFN, supports this notion. The molecular weight of PAF is approximately 2-3 X 10(4). Thus, the present studies suggest the requirement of at least two signals for the full maturation of macrophages, a cytokine represented by DF and a lymphokine, by PAF.