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Splenic T lymphocytes of rats treated with complete Freund's adjuvant (CFA) spontaneously release a lymphokine that inhibits the glycosylation of IgE-binding factors during their biosynthesis and provides the factors with biologic activity: selective suppression of the IgE response. The lymphokine, which is called glycosylation-inhibiting factor, also prevents IgE-induced increases in Fc epsilon R+ cells. The glycosylation inhibiting factor is formed by stimulation of BCG-primed spleen cells with PPD and participates in the selective formation of IgE-suppressive factors. The lymphokine is derived from OX 8+ T cells in both the CFA and BCG systems. The glycosylation-inhibiting factor is a 16,000-dalton peptide, as estimated by gel filtration, and specifically binds to monoclonal antibody against lipomodulin, a phospholipase inhibitory protein. Furthermore, "lipomodulin" was detected by radioimmunoassay in the 16,000-dalton fraction that contained glycosylation inhibiting factor. The fraction did not inhibit phospholipase A2 but after alkaline phosphatase treatment, the fraction did inhibit phospholipase A2. Furthermore, purified lipomodulin obtained from glucocorticoid-treated rabbit neutrophils had the same biologic activities as glycosylation inhibiting factors; i.e., it inhibited both protein glycosylation of IgE-binding factors and IgE-induced expression of Fc epsilon R. The results collectively indicate that glycosylation-inhibiting factor is a fragment of phosphorylated lipomodulin.

Incubation with rat IgE of rat mesenteric lymph node cells obtained 8 days after infection with Nippostrongylus brasiliensis (Nb) resulted in the formation of soluble factors with affinity for IgE, i.e., IgE-binding factors(s). The factors were derived from T lymphocytes and were able to suppress an in vitro IgE response without affecting the IgG response. The minimum concentration of rat IgE for the induction of factor formation was 0.3 to 1.0 microgram/ml. Gel filtration of culture filtrates of the IgE-containing culture identified 2 components with IgE-binding activity; one component had a m.w. of between 10,000 and 20,000, and another component had a m.w. of between 25,000 and 50,000. Both factors could be purified by binding to IgE-Sepharose followed by elution at acid pH. Among the two IgE-binding factors, the lower m.w. component had the ability to suppress selectively the IgE response. In contrast to IgE-potentiating factor, which also had affinity for IgE, the IgE-suppressive factor failed to bind to lentil lectin Sepharose. Formation of IgE-specific suppressive factor was not limited to the lymphocytes obtained 8 days after Nb-infection. Incubation with IgE of lymphocytes obtained 4 wk after infection resulted in the formation of both IgE-specific suppressive factor and IgE-potentiating factor, and the activity of the suppressive factor was greater than that of the potentiating factor.

Mesenteric lymph node (MLN) cells from rats treated with complete Freund's adjuvant (CFA) spontaneously released soluble factors, which have affinity for IgE (IgE-binding factors). The factors were purified by absorption of culture filtrates of the lymphocytes with IgE-Sepharose followed by elution at acid pH. Purified IgE-binding factors selectively suppressed IgE-forming cell response of DNP-ovalbumin-primed MLN cells to homologous antigen without affecting the IgG2 response. Fractionation of purified IgE-binding factors on lentil lectin-Sepharose showed that the majority of IgE-binding factors did not have affinity for lentil lectin, whereas a minor fraction was bound to the lectin-coupled beads. It was found that IgE-binding factors lacking affinity for lentil lectin suppressed IgE response, whereas those eluted from lentil lectin-Sepharose selectively enhanced IgE response. Serum of CFA-treated animals also contained both IgE-suppressive factor and IgE-potentiating factor, and the amount of the former factor was more than the latter. The source of IgE-suppressive factor is non-B lymphocytes. Depletion of Fc, receptor(+) lymphocytes in MLN cells from CFA-treated animals did not affect the formation of IgE-suppressive factor, indicating that Fc epsilon R-bearing cells are not the source of the factor.

Previous experiments have shown that Fc epsilon receptor-bearing (Fc epsilon R(+)) T lymphocytes in mesenteric lymph nodes (MLN) of rats infected with Nippostrongylus brasiliensis (Nb) release a soluble factor that selectively potentiates the IgE response. The IgE-potentiating factor has affinity for IgE, and can be detected by the ability to inhibit rosette formation of Fc epsilon R(+) cells with IgE-coated erythrocytes. The factor was bound to IgE-coated Sepharose and was eluted from the beads at acid pH. It was also found that the IgE-potentiating factor binds to lentil lectin-Sepharose, indicating that the factor is a glycoprotein. The affinity of the factor for IgE was lost after treatment with trypsin but was maintained after treatment with neuraminidase. However, the ability of the factor to potentiate the IgE response was lost after neuraminidase treatment. The results suggested that the factor's binding site for IgE is associated with a protein (peptide) moiety but that its carbohydrate moiety is essential for its biologic activity. When MLN cells were incubated at 37 degrees C, a substantial amount of the IgE-potentiating factor was released into culture medium within 4 hr even in the presence of cycloheximide. Pretreatment of the cells with trypsin, which removed Fc epsilon R, markedly diminished the release of IgE-potentiating factor, suggesting that the factor is derived from Fc epsilon R on the cell surface.

T lymphocytes in the mesenteric lymph nodes of rats infected with Nippostrongylus brasiliensis spontaneously released a soluble factor that selectively potentiated the IgE-forming cell response of antigen-primed cells to homologous antigen. The factor could enhance the IgE response of DNP-OA-primed cells to DNP-HSA and T cell-replacing factor. In contrast, the treatment of OA-primed T cells with the factor failed to enhance either the IgE or IgG response of the mixture of DNP-KLH primed cells and OA-primed T cells to DNP-OA. The results collectively suggested that the target cells of the IgE-potentiating factor are B cells. Indeed, IgE potentiating factor was absorbed by B cells rather than T cells or thymocytes. Evidence was obtained that IgE-potentiating factor could be absorbed by IgE-bearing B cells or IgE-coupled Sepharose, indicating that the factor had affinity for IgE. It appeared that the potentiating factor bound to IgE-bearing B cells and selectively enhanced the differentiation of IgE-B cells to IgE-forming cells. It was also found that the major source of the factor was Fc epsilon R-bearing T cells.

An i.p. injection of Bordetella pertussis vaccine (BP) into rats induced the formation of soluble factors that had affinity for IgE (IgE-binding factors). The factor was detected in the serum of BP-treated animals 5 to 7 days after the treatment. Their circulating lymphocytes as well as spleen cells spontaneously released IgE-binding factors in the serum of BP-treated rats and those released from their circulating lymphocytes had affinity for lentil lectin, and the ability to selectively potentiate an in vitro IgE response of DNP-OA primed cells to homologous antigen. The molecular size of IgE-potentiating factor was between 10,000 and 20,000, and was comparable to that formed by lymphocytes of rats infected with Nippostrongylus brasiliensis. Evidence was obtained that IgE-potentiating factor was derived from Fc epsilon R(+) T cells, with a T cell marker identified by monoclonal antibody W 3/25. Their production of IgE-potentiating factor may be the basis of the adjuvant effect of BP on the IgE response.

Incubation of rat lymphocytes with homologous IgE induced an increase in Fc epsilon R(+) lymphocytes and the formation of IgE-binding factors. Pretreatment of rat lymphocytes with 1 to 5 microM dexamethasone, however, prevented the IgE-induced expression of Fc epsilon R on both B and T lymphocytes. Upon incubation with IgE, T cells activated with 10 micrograms/ml Con A produced IgE-potentiating factors that had affinity for lentil lectin and selectively enhanced the IgE response. Pretreatment of the Con A-activated T cells with dexamethasone, before incubation with IgE, changed the nature of IgE-binding factors formed by the cells. The majority of IgE-binding factors formed by the dexamethasone-treated, Con A-activated cells failed to bind lentil lectin Sepharose and selectively suppressed the IgE response. An injection of 0.2 mg dexamethasone into rats infected with Nippostrongylus brasiliensis markedly diminished the proportion of Fc epsilon R(+) lymphocytes in their mesenteric lymph nodes. The lymph node cells from the infected animals spontaneously released IgE-potentiating factors in vitro. However, the majority of IgE-binding factors formed by the mesenteric lymph node cells from the dexamethasone-treated, Nb-infected animals lacked affinity for lentil lectin and selectively suppressed the IgE response. The results indicate that glucocorticoid treatment prevented the glycosylation of IgE-binding factors and thereby changed the biologic activities of the factors.

Differences between the conditions for an eosinophil chemotactic factor (ECF) and macrophage chemotactic factor (MCF) production by lymphoid cells of mesenteric lymph nodes and spleen were studied in guinea pigs. If lymphoid cells were washed less than 4 hr after concanavalin A (Con A) stimulation and were cultured for an additional 24 hr, they failed to produce ECF, whereas Con A stimulation for 1 hr before washing was sufficient to stimulate them to produce MCF. Subsequently, it was shown that heat-labile soluble factors (termed ECF-PF) with potentiating activity for ECF production are produced from macrophages by 5 micrograms/ml Con A activation. When ECF-PF were added to the cell culture with 5 micrograms/ml Con A, the lymphoid cells could produce ECF even when they were washed 2 hr after Con A stimulation and were cultured for an additional 24 hr, suggesting that ECF-PF plays a critical role in the early stage of ECF production. The lymphoid cells were also able to produce ECF even when they were cultured with ECF-PF and a suboptimal dose of Con A (1 microgram/ml) for ECF production. Protein synthesis seemed to be essential for ECF-PF production. The ECF-PF activity was associated with two separated molecular fractions with m.w. of about 50,000 to 70,000 and of 10,000 to 20,000. It is thus suggested that ECF is produced from T cells by Con A stimulation under conditions which differ, at least, from those for MCF in the requirement of ECF-PF.

Delayed tissue eosinophilia in DNP-ovalbumin-induced allergic inflammatory skin lesions of guinea pigs was markedly enhanced by previous treatment with alum hydroxy gel (Alum) or Bordetella pertussis vaccine. This enhancement seemed due to increased production of a lymphocyte-derived eosinophil chemotactic factor (ECF) at the skin site. Treatment of animals with Alum potentiated antigen-induced in vitro ECF production by lymphoid cells from spleen and mesenteric lymph node of sensitized animals. The co-culture supernatants of lymphoid cells from Alum-treated animals also potentiated concanavalin A (Con A)-induced in vitro ECF production. The potentiating effect of Alum on ECF production seemed to be ascribed to the release of soluble factors from macrophages of the Alum-treated animals. The macrophage-derived soluble factor ECF-potentiating factor (ECF-PF) selectively potentiated ECF production but not macrophage chemotactic lymphokine production by Con A-stimulated lymphoid cells from normal animals. ECF-PF activity was associated with two separate m.w. fractions: one was 50,000 to 70,000 and the other was 10,000 to 20,000. The present study provides one of the explanations for enhanced ECF production by adjuvants, such as Alum and Bordetella pertussis vaccine.

Attempts were made to induce the formation of soluble factors having affinity for IgE (IgE-binding factors) by activated T cells. Normal rat mesenteric lymph node (MLN) cells were cultured in 1 microgram/ml Con A for 48 hr or in 10 microgram/ml Con A for 72 hr, and Con A-activated lymphocytes were incubated with rat IgE. It was found that IgE induced an increase in the proportion of Fc epsilon R(+) cells in the Con A-activated cells and formation of IgE-binding factors. However, the nature of IgE-binding factors formed by the cells were different depending on the concentration of Con A for activation. Thus, IgE-binding factors formed by 10 microgram/ml Con A-activated cells had a high affinity for lentil lectin and enhanced the IgE-forming cell response of DNP-OA primed cells. In contrast, the majority of IgE-binding factors formed by 1 microgram/ml Con A-activated cells failed to bind to lentil lectin and suppressed the IgE response. Induction of Fc epsilon R by IgE on Con A-activated cells was prevented by tunicamycin, which inhibits protein glycosylation. This antibiotic did not prevent the IgE-induced formation of IgE-binding factors but changed the nature of the factors formed by 10 microgram/ml Con A-activated cells. The majority of IgE-binding factors formed in the presence of tunicamycin lacked affinity for lentil lectin and Con A, and suppressed, rather than enhanced, the IgE response. The nature and amount of IgE-binding factors formed by 1 microgram/ml Con A-activated cells was not affected by tunicamycin. The results suggest that glycosylation of IgE-binding factors during their biosynthesis is an important step in determining their biologic function.