IgE-binding factors from mouse T lymphocytes. I. Formation of IgE-binding factors by stimulation with homologous IgE and interferon

Incubation of normal mouse spleen cells with homologous IgE resulted in the formation of soluble factors that inhibited rosette formation of mouse Fc epsilon R+ cells with IgE-coated ox erythrocytes. The soluble factors could be absorbed with mouse or rat IgE coupled to Sepharose and recovered from the beads by acid elution. However, the factors had no affinity for either human IgE or mouse IgG. The IgE-binding factors were derived from T cells. Production of the factors required Lyt1+ T cells and Fc gamma R+ cells, which suggests that the factors are derived from Fc gamma R+ Lyt 1+ T cells. The molecular size of IgE-binding factors was approximately 15,000 daltons. When IgE-binding factors were formed by BALB/c spleen cells, nearly one-half of the factors had affinity for lentil lectin, and the remaining half of the factors failed to bind to the lectin. The proportion of the two species of IgE-binding factors differed depending on mouse strains. The majority of the factors formed by B6D2F1 spleen cells had affinity for lentil lectin, but those formed by SJL spleen cells failed to bind to the lectin. The IgE-binding factors were also induced by incubation of normal spleen cells with polyinosinic-polycytidylic acid (pI:pC). The nucleotide stimulated splenic adherent cells to form "inducers" of IgE-binding factors, which in turn induced normal lymphocytes to form IgE-binding factors. The inducers of IgE-binding factors were inactivated (or neutralized) by antibodies specific for mouse Type I interferon. It was also found that purified mouse beta interferon could induce the formation of IgE-binding factors. IgE-binding factors induced by pI:pC consisted of two different molecules: one had a m.w. of 15,000 daltons, and another had a m.w. of between 40,000 and 60,000 daltons.     

T-cell-derived IgE-binding factors. I. Cloned and transformed T cells producing IgE-binding factors

In order to derive T cell clones that can produce factors that specifically regulate the IgE response, Fc epsilon receptor (Fc epsilon R)-positive T cells were isolated from two patients with hyper-IgE states (either the tropical pulmonary eosinophilia syndrome or the hyper-IgE syndrome) and transformed using the human T cell leukemia/lymphoma virus. After infection, these T4-positive, Fc epsilon R-positive T cells manifested a fourfold increase in the surface interleukin 2 receptor, a 10-fold increase in Fc epsilon R, and acquired viral markers not present in the uninfected cells. Cloning soon after transformation allowed for the isolation of 17 distinct T cell clones producing IgE-binding factors. The levels of these binding factors released in supernatant fluid from these clones ranged from 10 to 98 U (negative less than 1 U) using a radioimmunoassay. Furthermore, the presence and kinetics of production of these binding factors also could be demonstrated by metabolic labeling studies. These cells should prove potent immunologic tools for dissecting the various regulatory mechanisms involved in IgE production both in the normal state and in pathologic conditions.     

Modulation by soluble factors of gelatinase activities released by osteoblastic cells

This study investigated the ability of normal human osteoblasts (hOb) and osteogenic sarcoma cells (MG-63 and SaOS2) to produce gelatinases and undergo modulation by interleukin 1beta (IL-1beta), interleukin 6 (IL-6), oncostatin M (OSM), leukaemia inhibitory factor (LIF), growth hormone (GH) and insulin-like growth factor-I (IGF-I). Gelatinase activities were determined by zymogaphy, and a quantitative analysis was performed by ELISA. The MMP-2 activities of the three cell lines were significantly increased in the presence of IL-1beta and IL-6, but no modulation of MMP-2 activities was observed in the presence of OSM, LIF and GH. IGF-I increased the activity released by SaOS2 and hOb, but no modulation was detectable in MG-63 cell conditioned medium. An upmodulation of pro-MMP-2 secretion by SaOS2 and hOb was observed for all soluble factors used, whereas an upmodulation of pro-MMP-2 secretion by MG-63 was observed only in the presence of IL-1beta, IL-6 and IGF-I. Thus, osteoblastic cells modulated by cytokines can be involved in bone resorption as a result of the protease activities released.     

Inhibition of ongoing myeloma IgE synthesis in vitro by activated human T cells

The ability of activated T cells to suppress ongoing IgE synthesis in vitro was assessed using U266--a human myeloma cell line spontaneously producing IgE. T cells were able to inhibit U266 IgE synthesis in the presence of 10 micrograms/ml of Con A by 41.8% (p less than 0.01). T cells preincubated with 10 or 50 micrograms/ml of Con A and washed extensively were still able to inhibit U266 IgE synthesis in the absence of Con A by 41 and 46% (p less than 0.05 and p less than 0.02, respectively). The decrease in IgE measured was due to inhibition of newly formed IgE by U266, as shown by control experiments with cycloheximide. The inhibition was not due to the simple depletion of nutrient growth factors by the activated T cells, as it did not occur with MOLT-4, T cells that are very active metabolically; nor could it be reversed with medium containing IL 2 and B cell growth factors. Culture supernatants of Con A-activated T cells were also able to suppress IgE synthesis by U266 (21%; p less than 0.01), which suggests that upon appropriate activation, T cells secrete material(s) with inhibitory properties for IgE synthesis. Activation of T cells by mixed lymphocyte culture using puromycin-treated lymphoblastoid cell lines as stimulators also generated T cells that had suppressive activity for IgE synthesis. T cells activated with Con A and subsequently incubated with IgE demonstrated a diminished ability to suppress IgE synthesis. This observation is in agreement with the finding that patients with high levels of IgE may lack isotype-specific suppressor T cells for spontaneous IgE secretion. However, T cells from such patients have so far shown variable loss of IgE suppressive function. These results suggest that human IgE synthesis is susceptible to inhibition at a very differentiated stage, and this may be important in expression of allergic diseases.     

IgE class-specific suppressor T cells and factors in humans

An in vitro experimental system for the induction of IgE production has been established with human peripheral blood lymphocytes (PBL). The stimulation of human PBL with PWM plus Cowan I induced IgM-, IgG-, and IgE- producing cells when assessed by reverse plaque assay. T cells, which had been isolated from patients with pulmonary tuberculosis and incubated with PPD plus IgE for 5 days, showed a suppressive effect on the polyclonal IgE response induced by PWM plus Cowan I. The direct addition of activated T cells, as well as the culture supernatant from activated T cells, abrogated the IgE response; the IgG response was not affected. The IgE-specific suppressive activity in the supernatant was specifically absorbed by an IgE column and could be eluted with acid buffer. The results demonstrated the presence of a human IgE binding factor(s), which showed its suppressive effect selectively in the IgE antibody response.     

Regulation of DNA synthesis and antibody production by soluble factors released by bone marrow cells

Bone marrow cells (BMC) suppressed the antibody response of spleen cells across a cell impermeable membrane. Fractionation of BMC supernatants by column chromatography and ultrafiltration revealed the presence of a suppressor factor and an enhancing factor which acted antagonistically. Bone marrow enhancing factor (B-EF) had a molecular weight greater than 20,000, enhanced antibody synthesis, and stimulated DNA synthesis in thymocytes but not BMC. Bone marrow suppressor factor (B-SF) was produced by non-adherent BMC, had a molecular weight 1000 to 10,000, suppressed the antibody response in vivo and in vitro, and stimulated DNA synthesis in BMC but not thymocytes. The possible role of these factors in homeostasis and regulation is discussed.     

Regulatory effects of human IgE-binding factors on the IgE response of rat lymphocytes

Normal human peripheral blood T cells were propagated in the presence of human interleukin 2, and activated cells were incubated with human IgE-dimer to induce IgE binding factor formation. The cells were then fused with a mutant of the human T cell line CEM. Five of the T cell hybridomas formed IgE binding factors upon incubation with human IgE-dimer. Because IgE binding factors formed by the human T cell hybridomas had affinity not only for human IgE but also for rat IgE, the biologic activities of the factors were evaluated by using antigen-primed rat mesenteric lymph node (MLN) cells. When parent T cells were propagated with crude IL 2, which contained glycosylation enhancing factor (GEF), IgE binding factors formed by all of the five hybridomas had affinity for Con A, but only a fraction of the factors bound to lentil lectin. The 60,000 and 15,000 IgE binding factors formed by two representative hybridomas, i.e., 166A2 and 166G11, selectively potentiated the IgE-forming cell response of rat MLN cells. When parent T cells were obtained by propagation with purified IL 2, which did not contain GEF, and the cells were incubated with IgE-dimer in the presence of glycosylation inhibiting factor (GIF), T cell hybridomas constructed from the cells formed IgE binding factors that lacked affinity for Con A but bound to peanut agglutinin (PNA). The 30,000 IgE binding factors formed by two of such hybridomas, 398A3 and 400G2, selectively suppressed the IgE response of rat MLN cells. It was also found that the biologic activities and carbohydrate moieties of human IgE binding factors could be switched by changing the culture conditions of the hybridomas. When the 166A2 hybridoma was cultured with human IgE in the presence of bradykinin, essentially all of the IgE binding factors that were formed by the cells bound to lentil lectin, and the factors that were formed in the presence of bradykinin exerted higher potentiating activity than those obtained in the absence of bradykinin. On the other hand, IgE binding factors formed by the same cells in the presence of GIF had affinity for PNA, and selectively suppressed the IgE response of rat MLN cells.     

Induction of IgG and IgE synthesis in normal B cells by autoreactive T cell clones

During the course of generating tetanus toxoid (TT)-specific T cell clones frm an HLA-DR2,7 donor, four clones were obtained which proliferated in the presence of autologous monocytes alone without the addition of TT antigen. This proliferation was specifically inhibited by anti-HLA-DR framework mouse monoclonal antibody, and appeared to be HLA-DR-restricted. Two of the clones proliferated in response to HLA-DR2-bearing monocytes, and the other two clones proliferated in response to HLA-DR7-bearing monocytes. The capacity of these four autoreactive human T cell helper clones to induce IgE synthesis in B cells was studied. All four clones stimulated autologous peripheral blood B cells to synthesize IgE and IgG antibody. Induction of IgE synthesis in B cells by the autoreactive T cell clones followed the same pattern of HLA-DR restriction which governed the proliferative response of these clones. These results suggest that the interaction of autoreactive helper T cells with B cell HLA-DR antigens may be important in the activation of IgE immune responses in humans.     

The regulation of protein synthesis in animal cells by serum factors.

We have investigated the regulation of protein synthesis in animal cells by serum factors. Withdrawal of serum from the medium of actively dividing Vero cells resulted in an immediate decline in the rate of peptide chain elongation (Hassell and Engelhardt, 1973). Assay of elongation factor I (EFI) activity in the post-ribosomal supernatant as well as that associated with the ribosomes revealed that serum deprivation resulted also in reduction in the activity of this factor. The decline in the activity of EFI after serum deprivation occurred to the same extent and at the same time as the decline in the in vivo rate of protein synthesis and the in vitro peptide synthetic capacity of cell-free extracts. A temporal correlation therefore exists among the in vivo rate of protein synthesis, the peptide synthetic activity of cell-free extracts, and the activity of EFI. The activity of peptidyl transferase was not altered by serum deprivation. The loss of extract peptide synthetic activity resulting from serum deprivation was reversible since serum addition to previously serum-starved cultures resulted in full restoration of activity for polyphenylalanine (polyPhe) synthesis within 3 h. Moreover, RNA synthesis was not required for this turn-on of polyPhe synthesis. Vased on these data we conclude that a translational control mechanism is operative in Vero cells deprived of serum.     

Induction of the differentiation of memory T killer cells with factors released from macrophage-like cell lines

Macrophage-like cell lines J774.1 and WEHI-3 as well as peritoneal exudate macrophages have been demonstrated to produce factors which induce the differentiation of memory cells into specific T killer cells in the absence of an added antigen. LPS stimulation was required for J774.1 cells and peritoneal macrophages to produce the factors but not for WEHI-3 cells. Interferon seemed to be one of the responsible factors. However, macrophages seem to produce other active factors; one has a molecular weight (MW) of more than 80,000 and lacks thymocyte mitogenic activity; another, with a weak thymocyte mitogenic activity, has a MW of 38,000 to 44,000. The low MW thymocyte mitogenic factor (interleukin-1) showed weak T killer cell differentiating activity.     

Growth factors are released by mechanically wounded endothelial cells

Growth factors may be required at sites of mechanical injury and normal wear and tear in vivo, suggesting that the direct action of mechanical forces on cells could lead to growth factor release. Scraping of cells from the tissue culture substratum at 37 degrees C was used to test this possibility. We show that scraping closely mimics in vitro both the transient plasma membrane wounds observed in cells subject to mechanical forces in vivo (McNeil, P. L., and S. Ito. 1989. Gastroenterology. 96:1238-1248) and the transient plasma membrane wounds shown here to occur in endothelial cells under normal culturing conditions. Scraping of endothelial cells from the culturing substratum released into the culture medium a potent growth-promoting activity for Swiss 3T3 fibroblasts. Growth-promoting activity was released rapidly (within 5 min) after scraping but was not subsequently degraded by the endothelial cells for at least 24 h thereafter. A greater quantity of growth-promoting activity was released by cells scraped 4 h after plating than by those scraped 4 or 7 d afterwards. Thus release is not due to scraping-induced disruption of extracellular matrix. Release was only partially cold inhibitable, was poorly correlated with the level of cell death induced by scraping, and did not occur when cells were killed with metabolic poisons. These results suggest that mechanical disruption of plasma membrane, either transient or permanent, is the essential event leading to release. A basic fibroblast growth factor-like molecule and not platelet-derived growth factor appears to be partially responsible for the growth-promoting activity. We conclude that one biologically relevant route of release of basic fibroblast growth factor, a molecule which lacks the signal peptide sequence for transport into the endoplasmic reticulum, could be directly through mechanically induced membrane disruptions of endothelial cells growing in vivo and in vitro.     

IgE-binding factors from mouse T lymphocytes. III. Role of antigen-specific suppressor T cells in the formation of IgE-suppressive factor

BDF1 mice were given three i.v. injections of ovalbumin (OA) to induce antigen-specific suppressor T cells. Incubation of spleen cells of OA-treated mice with homologous antigen resulted in the formation of IgE-suppressive factor. This factor was not derived from antigen-specific suppressor T cells, but suppressor T cells were essential for determining the nature of IgE-binding factors formed. In the spleen cells of OA-treated mice, antigenic stimulation of antigen-primed Lyt-1+ (helper) T cells resulted in the formation of inducers of IgE-binding factor, whereas Lyt-2+, I-J+ T cells released glycosylation-inhibiting factor (GIF), and these two factors, in combination, induced unprimed Lyt-1+ T cells to form IgE-suppressive factor. The role of GIF is to inhibit the assembly of N-linked oligosaccharides on IgE-binding factors during their biosynthesis, and thereby provide them with a biologic activity: suppression of the IgE response. Under the experimental conditions employed, GIF was released spontaneously from antigen-specific suppressor T cells. However, antigenic stimulation of the cells enhanced the release of the factor. GIF from antigen-specific suppressor T cells has a m.w. of 25,000 to 30,000, as estimated by using gel filtration, binds to anti-I-J alloantibodies and to a monoclonal antibody specific for lipomodulin, and has affinity for specific antigen. The possible relationship between antigen-specific GIF and antigen-specific suppressor factors is discussed.     

Frequency analysis of IgE-secreting B lymphocytes in persons with normal or elevated serum IgE levels

The immunoregulatory mechanisms that determine the high serum IgE antibody levels in disorders such as helminth parasite infections and the hyper-IgE recurrent infection syndrome (HIE) remain poorly understood. To assess whether elevated serum IgE levels result from an increased number of B lymphocytes committed to IgE production, the proportion of IgE-producing B lymphocytes was determined by a filter immunoplaque assay using PBMC from persons with a broad range of serum IgE levels that included normal persons (n = 9) and patients with loiasis (n = 12), tropical pulmonary eosinophilia (TPE) (n = 6), lymphatic filariasis (n = 28), and HIE (n = 8). PBMC from these persons were assessed for production of in vitro IgE. The geometric mean number of IgE-secreting cells in 10(5) B lymphocytes in PBMC was 0.42 (range 0-2.2) in normal persons, 5.6 (range 0.1-35.5) among patients with loiasis, 9.4 (range 0-53.2) among patients with lymphatic filariasis, 52 (range 31.5-115) among patients with TPE, and 218 (range 56-1404) among patients with HIE. When all study subjects were grouped, there were significant correlations with serum IgE levels (r2 = 0.78; p less than 0.0001) and net spontaneous in vitro IgE production (r2 = 0.8; p less than 0.0001). Estimates of the amount of IgE production per B lymphocyte were similar among normal persons, patients with filarial infections, and patients with TPE (geometric means of 134, 96, and 141 pg/ml/cell, respectively); in contrast, for HIE patients, IgE production by individual B cells was significantly lower (geometric mean 28 pg/ml/cell; p less than 0.001). These observations demonstrate that clonal expansion of IgE-producing B lymphocytes is a major mechanism underlying the elevated serum IgE levels seen in persons with hyper-IgE states.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

Modulation of immune responses by suppressor T cells.

The activity of suppressor T cells has been demonstrated in almost every phase of the immune response. These regulatory cells modulate both humoral and cell-mediated immunity utilizing antigen-specific and nonspecific mechanisms. For comparative purposes two murine models are described, the nonspecific suppressor T cell stimulated by the mitogen concanavalin A and the antigen-specific suppressor T cell stimulated by injection of the synthetic terpolymer acid 60-L-alanine30-L-tyrosine10 (GAT) in nonresponder mice. These two T cells are similar to expression of Ly alloantigens, ability to inhibit antibody responses, and the mediation of suppression, at least in part, by soluble products. However, differences in radio-resistance and antigenic specificity of the suppressor T cells, as well as differences in molecular characteristics of the soluble factors and their targets suggest that these T cells regulate the immune response by different mechanisms. The relationship of these two suppressor T cells to other nonspecific and antigen-specific suppressor T cells is discussed.     

Soluble factors released by activated cytotoxic T lymphocytes interfere with death receptor pathways in neuroblastoma

Neuroblastoma (NB) is often described as an unfavorable target for both HLA-restricted and death receptor-mediated elimination by cytotoxic T lymphocytes (CTLs) due to low or absent HLA class I and caspase-8 expression. We investigated the effects of soluble factors released by CTLs activated by TCR triggering (named as activated supernatant; AS) on the levels and composition of cell surface molecules involved in HLA-restricted and HLA-independent NB cell recognition (surface immune phenotype). Using a panel of long-term propagated NB cell lines and freshly isolated primary human NB cells, we analyzed surface expression of the (1) cognate receptors for TNFα, Fas and TRAIL; (2) HLA class I and II heterodimers; (3) adhesion molecules; (4) the intracellular expression and activation of caspase-8, as well as (5) the susceptibility of NB cells to death receptor-mediated killing prior to and after exposure to AS. The exposure of NB cells to soluble factors released by activated CTLs skewed the surface immune phenotype of both long term cultured and primary NB cells, induced the expression and activation of caspase-8 and increased the susceptibility of tumor cells to lysis by TRAIL and Fas-agonistic antibody. Blocking experiments identified IFNγ and TNFα as main factors responsible for modulating the surface antigens of NB cells by AS. Our data suggest that recruitment of CTLs activated on third party targets into the vicinity of the NB tumor mass, may override the “silent” immune phenotype of NB cells via the action of soluble factors. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by grants from the Swedish Children’s Cancer Foundation, the Swedish Cancer Society, the Cancer Society of Stockholm, the King Gustav the Vth Jubilee Fund, Karolinska Institutet and the Swedish Research Council.     

Inhibition of interleukin 2 production by factors released from tumor cells

In previous studies, cultured melanoma cells were shown to have a suppressive influence on the induction of cytotoxic T cells. Our investigation of the mechanism of these effects revealed that supernatants from certain cultures of melanoma cells contained inhibitory activity against the production of interleukin 2 (IL 2) from phytohemagglutinin (PHA)-stimulated cultures of lymphocytes. These supernatants did not inhibit interleukin 1 production, and also did not inhibit the mitogenic activity of performed IL 2 on IL 2-dependent target cells. Production of the inhibitory activity could be reduced by inhibitors of protein synthesis, but this activity was not inhibited by digestion with the proteolytic enzymes trypsin or pronase. Gel filtration analysis of tumor supernatants revealed that the majority of the inhibitory activity was detected in fractions of approximately 44 and 7 Kd. The addition of supernatants with inhibitory activity to PHA-stimulated cultures of lymphocytes was associated with reduced transition of cells from G1 to S phase of cell division, which could be reversed by the addition of IL 2. Preliminary studies suggest that the release of the factor(s) from melanoma cells may be related to rapid progression of tumor growth in patients, and therefore may be of prognostic significance in tumor host relationships.     

A dialyzable protein synthesis inhibitor released by mammalian cells in vitro

Macrophages, lymphocytes, and HeLa cells when incubated $\text{in vitro}$ at 37° in Krebs-Ringer phosphate buffer release a dialyzable, heat-stable, ninhydrin-reacting factor which inhibits protein synthesis by intact cells and isolated ribosomes. Release of the inhibitor appears to be dependent on metabolism. Partial purification of the inhibitor by Sephadex G-10 chromatography suggests it has a molecular weight of 400–600.