Inhibition of kit expression in P815 mouse mastocytoma cells by a hammerhead ribozyme.

Using P815 cells, we designed and tested an antisense hammerhead ribozyme for its ability to mediate cleavage of mouse c-kit mRNA at the transmembrane-encoding region. The ribozyme demonstrated in vitro cis cleavage of extraneous 5' sequence in the ribozyme construct as well as trans cleavage of a target c-kit mRNA sequence. The in vitro cleavage was effective at 37 degrees C. A subpopulation of P815 cells transfected with the pCDNA1 plasmid-containing the anti-c-kit ribozyme demonstrated inhibited Kit expression as indicated by flow cytometry, but the inhibition could not be maintained over time. The anti-c-kit ribozyme was put under the control of a tetracycline-inducible two-plasmid system of expression. Incomplete inhibition of Kit expression was observed when transcription of the ribozyme was allowed by the absence of tetracycline, but not when tetracycline was present. Finally, complete inhibition of Kit expression was observed when pCDNA1-ribozyme-transfected cells were analyzed immediately after enrichment by cotransfection with a plasmid expressing green fluorescent protein, followed by cell sorting. The results suggest that anti-c-kit ribozymes might be useful for inhibiting Kit expression in hyperplastic mast cells, if delivery of ribozymes can be optimized.

Placental Fas ligand expression is a mechanism for maternal immune tolerance to the fetus.

Fas ligand (FasL) is a peptide that plays an important immunoregulatory role in limiting the host immune response. Several studies have shown that the expression of FasL in the anterior chamber of the eye and the testis allows these tissues to be immunoprivileged sites. Immunotolerance is achieved by binding of FasL to its receptor (Fas) on activated immune cells, which results in cell apoptosis. To determine whether FasL has a role in maternal immune tolerance to the fetus, we looked for the expression of FasL in the human placenta. Immunoperoxidase staining localized FasL to both syncytiotrophoblast and cytotrophoblast in placental villi and chorionic extravillous trophoblast. Western analysis demonstrated FasL in placental villi and a human first-trimester trophoblast cell line (ED27). In contrast, Fas was colocalized to CD45 (leukocyte common antigen) positive cells found in maternal decidua. When isolated peripheral blood lymphocytes were induced to express Fas with phytohemagglutinin (PHA) and interleukin-2 (IL-2) and then cocultured with trophoblast, 30% of the lymphocytes underwent apoptosis, as determined by the in situ death (TUNEL) assay. Neutralizing antibodies to FasL inhibited apoptosis by 40% in these studies. In contrast, activated lymphocytes cocultured with non-FasL-expressing fibroblasts or unactivated non-Fas-expressing lymphocytes cocultured with ED27 trophoblast showed little evidence of apoptosis. These findings suggest that FasL expressed by fetal trophoblast cells can induce apoptosis in activated lymphocytes there by providing a mechanism for maternal immune tolerance to the fetus.

IL-4 inhibits mouse mast cell Fc epsilonRI expression through a STAT6-dependent mechanism.

Mast cell activation by IgE-mediated stimuli is a central event in atopic disease. The regulation of the mast cell high affinity receptor, Fc epsilonRI, is poorly understood. We show that IL-4 can inhibit Fc epsilonRI expression on mouse bone marrow-derived mast cells and fetal liver-derived mast cell progenitors. This effect could be observed at 2.5 ng/ml IL-4 and was dose dependent. IL-4-mediated inhibition of cultured BMMC required 4 days of stimulation and was sustained at maximum levels for at least 21 days. The inhibition of Fc epsilonRI expression resulted in decreased sensitivity to IgE-mediated stimulation, as measured by serotonin release, and the induction of mRNA for IL-4, IL-5, IL-6, and IL-13. Additionally, IL-4 could abrogate the IgE-mediated increase in Fc epsilonRI expression. Lastly, IL-4-mediated inhibition was dependent upon expression of the STAT6 transcription factor, as STAT6-deficient bone marrow-derived mast cells did not decrease Fc epsilonRI levels in response to IL-4. These data argue for a homeostatic role of IL-4 in the regulation of Fc epsilonRI expression, a role that could be critical to understanding atopic disease.

Stem cell factor activates STAT-5 DNA binding in IL-3-derived bone marrow mast cells.

The Kit tyrosine kinase regulates growth and differentiation of many hematopoietic cells through a signal transduction process that remains to be fully elucidated. Kit has been shown to associate with the receptor for erythropoietin (Epo), which is known to activate the signal transducer and activator of transcription, STAT-5. To determine if Kit signal transduction activated latent DNA-binding factors, including STAT-5, we performed electrophoretic mobility shift assays on stem cell factor (SCF)-stimulated mouse bone marrow-derived mast cells (BMMCs). SCF led to the rapid and transient activation of a DNA-binding factor that was identified by supershift analysis as STAT-5. STAT-5 DNA binding was shown to be specific for the oligonucleotide of the correct sequence and was dose-responsive. Epo stimulation of BMMCs led to the activation of a DNA-binding activity that comigrated with the SCF-induced band, but peaked and was maintained at later time points than SCF-induced activation. These data indicate that SCF stimulation of Kit leads to activation of STAT-5 DNA binding with kinetics distinct from Epo-mediated stimulation.

Differential responsiveness of purified mouse c-kit+ mast cells and their progenitors to IL-3 and stem cell factor.

Murine mast cell development is regulated by a number of T cell- and fibroblast-derived growth factors, of which IL-3 and stem cell factor (SCF) appear to be the most important. To determine the relative effects of these two growth factors on different stages of developing mast cells, we isolated highly purified populations of c-kit+ mast cells and their progenitors and examined their ability to respond to SCF and/or IL-3. We show that purified c-kit+ cells isolated from bone marrow, mesenteric lymph nodes of Nippostrongylus brasiliensis-infected mice, and the peritoneal cavity do not form mast cell colonies in response to SCF as a single agent. Although unpurified populations of lymph nodes of Nippostrongylus brasiliensis-infected mice or peritoneal cells were able to form mast cell colonies in response to SCF alone, this probably reflects paracrine factors produced by accessory cells. IL-3 alone did not promote mast cell colony formation from either population. Within bone marrow, we detected both multipotential and unipotential mast cell progenitors. Interestingly, IL-3 alone promoted only the development of multipotential progenitors, while SCF plus IL-3 promoted both multipotential and unipotential mast cell progenitors. Together, these results indicate that the developmental/proliferative effects observed with a particular mast cell population is determined in part by the stage of maturation of mast cells at the time they are exposed to SCF and IL-3, and suggest that SCF primarily influences mast cell development only in the context of other cytokines.

Murine KIT+ lineage- bone marrow progenitors express Fc gamma-RII but do not express Fc epsilon-RI until mast cell granule formation.

We examined the expression of Fc epsilon-RI and Fc gamma-RII/III on mouse bone marrow cells enriched for hematopoietic progenitors including mast cell progenitors. Bone marrow cells were depleted of mature hematopoietic lineages and a primitive population of cells that express the proto-oncogene c-kit (KIT+ lineage- cells) was isolated. KIT+ lineage- cells stain positively using the Ab 2.4G2, indicating surface expression of Fc gamma-RII and/or Fc gamma-RIII. Fluorescent staining of intracytoplasmic domains of Fc gamma-RII and Fc gamma-RIII revealed that these cells express primarily Fc gamma-RII on their surface. KIT+ lineage- cells did express Fc gamma RIII alpha-chain protein, but predominately in the nuclear/perinuclear area. We could not detect surface expression of Fc epsilon-RI by KIT+ lineage- cells, although a heterogeneous population of KIT- cells does bind IgE with high affinity and may reflect cells of the basophilic lineage. KIT+ lineage- cells cultured with SCF and IL-3 generate numerous mast cells, whereas equivalent numbers of KIT- cells or naive bone marrow cells do not. In these cultures, surface expression of Fc epsilon-RI is detected on a small number of cells by day 3 of culture with increased surface expression levels correlating roughly with metachromatic granule formation. The fact that Fc gamma-RIII and Fc epsilon-RI are not expressed on the cell surface of KIT+ lineage- cells but appear later in hematopoietic development makes it unlikely that these receptors influence early hematopoietic differentiation. The role that might justify such a complete surface expression of Fc gamma-RII by bone marrow progenitors remains to be identified.

Role for the stem cell factor/KIT complex in Schwann cell neoplasia and mast cell proliferation associated with neurofibromatosis.

Schwann cells are the primary cell type in the disfiguring lesions associated with neurofibromatosis type 1 (NF-1). These lesions also contain abnormally high numbers of mast cells, a cell type which develops in response to stem cell factor. We report here that neonatal and adult rat and human Schwann cells, as well as a transfected rat Schwann cell line and a human Schwannoma line derived from an NF-1 patient, all produced stem cell factor mRNA and protein. In coculture experiments, surface expression of stem cell factor by neonatal rat Schwann cells was profoundly downregulated by contact with dorsal root ganglion neurites. The receptor for stem cell factor, KIT, was not expressed in normal Schwann cells but was expressed in the human Schwannoma line, suggesting that aberrant KIT expression may form an autocrine loop in certain Schwann cell neoplasias.

Expression of c-kit by mesenteric lymph node cells from Nippostrongylus brasiliensis-infected mice and by mast cell colonies developing from these cells in response to 3T3 fibroblast-conditioned medium.

Mast cell committed progenitors are nongranulated cells found in mesenteric lymph nodes of mice infected with Nippostrongylus brasiliensis (Nb-MLN) but not from normal mice. Mast cell committed progenitors can respond to either IL-3 or to a factor(s) present in 3T3 fibroblast conditioned media (F-CM) by formation of mast cell colonies. Previous studies from ours and other laboratories suggested that mast cell differentiation involved the W allele product, c-kit, as a receptor and Sl allele product, stem cell factor, as a growth factor. We report here that Nb-MLN cells, which can respond to F-CM by mast cell colony formation, also contain cells that express message for c-kit, and that c-kit message cannot be detected in naive mesenteric lymph node cells, which cannot respond to F-CM. Antisense oligonucleotides to c-kit inhibit mast cell colony formation by Nb-MLN cells in response to F-CM, but not to conditioned medium of PWM-stimulated spleen cells as a source of IL-3. The antisense oligonucleotides also inhibit the degree of granulation by mast cells derived from culture. The results suggest that c-kit and its ligand, stem cell factor, are necessary for mast cell-committed progenitors to proliferate and granulate in response to F-CM but not IL-3.

Murine follicular dendritic cells and low affinity Fc receptors for IgE (Fc epsilon RII).

The present study was undertaken to determine whether mouse follicular dendritic cells (FDC) bear Fc epsilon RII (CD23) and whether IgE-immune complexes are retained by FDC. Mouse Fc epsilon RII was localized by both L and electron microscopy using the mAb B3B4. In lymph nodes of normal mice, Fc epsilon RII was low but detectable on FDC. By 14 days after Nippostrongylus brasiliensis infection, the level of Fc epsilon RII increased on B lymphocytes located in the cortex of draining mesenteric lymph nodes. However, the Fc epsilon RII level on FDC remained low. Although numerous IgE-producing plasma cells were seen at day 14, very little IgE was associated with FDC. By 26 days after infection, Fc epsilon RII was observed on FDC in increased levels and IgE binding was clearly associated with FDC. Unexpectedly, FDC of control mice immunized with albumin in CFA to elicit an IgG response showed intense labeling for Fc epsilon RII. In contrast, the B cells exhibited very little Fc epsilon RII. IgE immune complexes were observed in association with FDC in the CFA-immunized mice. When mice were given a hapten-specific monoclonal of the IgE isotype, hapten carrier complexes were trapped and retained on Fc epsilon RII-bearing FDC. In conclusion, FDC were clearly one of the major murine cell types bearing Fc epsilon RII. IgE immune complexes were found in association with FDC and Fc epsilon RII appeared to play a major role in trapping and retaining IgE immune complexes. FDC Fc epsilon RII was subject to regulatory control, but the Fc epsilon RII level on FDC was regulated very differently from the Fc epsilon RII level on B cells.

c-kit expression by CD34+ bone marrow progenitors and inhibition of response to recombinant human interleukin-3 following exposure to c-kit antisense oligonucleotides.

The c-kit proto-oncogene encodes a receptor having tyrosine-specific kinase activity and has been mapped to chromosome 4 in the human and chromosome 5 in the mouse, at the dominant white spotting locus (W). Mutations at the W locus affect various aspects of murine hematopoiesis. The c-kit proto-oncogene has been shown to be expressed by leukemic myeloblasts, but not by normal unseparated human bone marrow cells. The role of this oncogene in differentiation and proliferation of human hematopoietic progenitors is presently undefined. To determine c-kit expression by normal hematopoietic progenitors, CD34+ cells were isolated from disease-free human bone marrow, and RNA-based polymerase chain reaction (PCR) techniques were used to assess expression. By this method, we have demonstrated c-kit expression by CD34+ bone marrow progenitors. To address the functional requirement for c-kit expression in normal human hematopoiesis, CD34+ cells were incubated in the presence of sense, antisense, or missense oligonucleotides to c-kit, and subsequently cultured in the presence of either recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or recombinant human interleukin-3 (rhIL-3). Exposure of CD34+ cells to c-kit antisense oligonucleotides significantly inhibited colony-forming ability of cells cultured in the presence of rhIL-3, but had no effect on colony formation of cells cultured in rhGM-CSF. Together, these data suggest a possible role for c-kit in hematopoietic proliferation and differentiation that may be linked to some, but not all, stimulatory factors.

The mast cell-committed progenitor. In vitro generation of committed progenitors from bone marrow.

Mast cell-committed progenitors are detected in the unique microenvironment of the mesenteric lymph node (MLN) of Nippostrongylus brasiliensis-infected mice but not in naive bone marrow. We have determined that MLN cells, after infection, produce high levels of IL-3, IL-4, and IgE, presumably in the form of immune complexes with antigens produced by the infecting helminth. After N. brasiliensis infection, peak production of these factors occurs several days before the peak appearance of mast cell-committed progenitors in the MLN. To determine if these factors play a role in mast cell commitment, we recreated these conditions, in vitro. Naive bone marrow cells were cultured with combinations of IL-3, IL-4, and IgE immune complexes, or on IgE-coated plates, and then assayed for acquisition of the ability to form mast cell colonies when supplemented with fibroblast-conditioned medium alone. IL-3 and IgE immune complexes, and, unexpectedly, IgE immune complexes alone were found to be capable of producing mast cell-committed progenitors, i.e., cells responsive to fibroblast-conditioned medium alone, from bone marrow, whereas IL-4 did not enhance production of mast cell-committed progenitors from bone marrow. Production of IFN-gamma peaked at the same time point as committed progenitor activity and may be responsible for down regulating the response.

The mast cell-committed progenitor. I. Description of a cell capable of IL-3-independent proliferation and differentiation without contact with fibroblasts.

We have identified a late, committed stage in the differentiation of the mast cell progenitor just before granulation. Mast cell committed progenitors (MCCP) are nongranulated cells with a density of 1.060 to 1.070 g/ml which can be harvested from the mesenteric lymph node of mice infected with Nippostrongylus brasiliensis. Mast cell-committed progenitors are able to proliferate and differentiate in the absence of IL-3 or IL-4 when cultured on a monolayer of embryonic skin or 3T3 fibroblasts and can form colonies in methylcellulose supplemented with fibroblast conditioned medium. Fibroblast conditioned medium appears to contain a soluble MCCP proliferation factor that maintains biologic activity when heated to 56 degrees C for 45 min but is destroyed by incubation with either trypsin or chymotrypsin. It can be selectively precipitated with 60 to 70% saturated ammonium sulfate. The factor is not absorbed by immobilized antibodies to nerve growth factor. The MCCP proliferation activity of the factor could not be mimicked by IL-1, IL-2, IL-4, granulocyte-macrophage-CSF, granulocyte-CSF, macrophage-CSF, IFN-alpha/beta, IFN-gamma, nerve growth factor, epidermal growth factor, serum fibronectin, heparin, or a number of glycosaminoglycans. At high salt concentrations, the factor passes through a 50-kDa membrane and can be concentrated above a 5-kDa membrane. MCCP acquire a connective tissue phenotype when cultured on a fibroblast monolayer and a mucosal phenotype when cloned in the presence of conditioned medium from PWM-stimulated spleen cells. When cultured in the absence of IL-3 on a monolayer of embryonic skin or 3T3 fibroblasts, mast cell-committed progenitors produce mast cells which stain with berberine sulfate suggesting a connective tissue phenotype; however, the mast cells that develop when mast cell-committed progenitors are cultured in the presence of IL-3 or conditioned media from PWM-stimulated spleen cells do not stain with berberine sulfate. MCCP intercalate into monolayers of embryonic skin or 3T3 fibroblasts, but T cells are not able to associate with the monolayer and can be completely washed away. Attempts to enrich mast cell-committed progenitors by intercalation and elution from embryonic skin monolayers proved unsuccessful, but some enrichment of mast cell-committed progenitors could be achieved by discontinuous Percoll gradients. Thus, we have identified a way to obtain late-stage, mast cell-committed progenitors in an environment that is virtually uncontaminated with other hematopoietic progenitors.

The mast cell-committed progenitor. II. W/Wv mice do not make mast cell-committed progenitors and S1/S1d fibroblasts do not support development of normal mast cell-committed progenitors.

We have previously reported that the population of mesenteric lymph node cells from normal BALB/c mice infected 14 days with the rodent nematode Nippostrongylus brasiliensis (Nb-MLN) contains a nongranulated mast cell-committed progenitor (MCCP) which does not require IL-3 for proliferation and differentiation if either a fibroblast monolayer or soluble factors produced by monolayers of 3T3 fibroblasts or embryonic skin are present in the culture. When Nb-MLN were cloned in a methylcellulose culture system using fibroblast conditioned medium as the only source of growth factors, numerous colonies of pure mast cells developed. We wished to determine whether the mast cell deficiency of W/Wv or S1/S1d mice could be explained by the failure of these mice to make either the MCCP or the factor to support proliferation and differentiation of the MCCP. We found that Nb-MLN from W/Wv mice were only able to produce mast cell colonies in response to a source of IL-3 such as conditioned medium from pokeweed mitogen-stimulated spleen cells (CM), and cultures given fibroblast conditioned medium as the only source of growth factors did not produce mast cell colonies. In contrast, Nb-MLN from mast cell deficient S1/S1d mice developed many mast cell colonies in methylcellulose cultures supplemented with either fibroblast conditioned medium or conditioned medium from PWM-stimulated spleen cells. These data suggest that S1/S1d mice but not W/Wv mice produce the mast cell progenitor that responds to fibroblast conditioned medium. To determine if mast cell deficient mice make the fibroblast derived factors that support development of the MCCP, monolayers were prepared from skin connective tissues of S1/S1d and W/Wv mice and Nb-MLN from normal BALB/c mice were cloned in the presence of conditioned medium from these monolayers. Fibroblast conditioned medium from monolayers prepared from W/Wv but not S1/S1d mice supported development of numerous mast cell colonies. Taken together, these data demonstrate that W/Wv mice are incapable of producing normal MCCP whereas S1/S1d fibroblasts fail to produce the appropriate factor to support the MCCP. In accordance with these data, a candidate for the gene product of each of these mutant alleles is discussed.

Mast cell differentiation in cultures of T cell-depleted mesenteric lymph node cells from Nippostrongylus brasiliensis-infected mice.

Lymphoid and bone marrow cells from normal and horse serum-immunized mice and lymphoid cells from Nippostrongylus brasiliensis-infected mice were cultured on monolayers of embryonic skin fibroblasts to analyze the factors which regulate the differentiation and proliferation of mast cells in vitro. Our results indicate that T cells can regulate the development of mast cells in vitro by either enhancement or suppression. In cultures of horse serum-immune spleen cells, inducer T cells are required for mast cells to develop. However, in cultures of mesenteric lymph node cells from N. brasiliensis-infected mice, inducer T cells are not required for mast cell development. This suggests that the development of mast cells may occur at discrete interleukin-3 (IL-3)-dependent and IL-3-independent stages. Mast cell precursors in the mesenteric lymph nodes of N. brasiliensis-infected mice may have already been acted on by inducer cells in vivo to become mast cell committed. While IL-3 does not appear to be required for mast cell development, these precursors do require the presence of a connective tissue microenvironment such as embryonic skin. The precursors can be inhibited from development by interferon preparations.

Monoclonal antibody specific for T cell-derived human IgE binding factors.

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.

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.

cDNA clones encoding IgE-binding factors from a rat-mouse T-cell hybridoma.

cDNA clones encoding rodent IgE-binding factors (IgE-BF) were isolated from cDNA libraries of a rat-mouse T hybridoma that secretes IgE-suppressive factor (IgE-SF) upon incubation with rat IgE. COS7 cells transfected with two of the cDNAs expressed IgE-BF, which selectively potentiate an in vitro IgE response. IgE-BF expressed in COS7 cells are glycoproteins of approximately equal to 60 and approximately equal to 11 kDa. DNA sequence analysis of an IgE-BF cDNA revealed a 556-amino acid (62 kDa) protein coding region. The results suggest that IgE-potentiating and IgE-suppressive factors share common precursor polypeptides and that the 11-kDa IgE-BF is derived from a 60-kDa precursor.

IgE-binding factors. Selective regulation of the IgE response by T cell factors.

Gene cloning of rodent IgE-binding factors was accomplished, using messenger RNA of a rat T cell hybridoma, 23B6, which produce IgE-suppressive factor. Transfection of cos 7 monkey kidney cells with a cDNA clone resulted in the formation of rodent IgE-potentiating factors. The results provided a definitive evidence that IgE-binding factors represent a single peptide chain, and that the IgE-potentiating factor and IgE-suppressive factor share a common structural gene. Nucleotide sequence of a cDNA provided predicted amino acid sequence of the 60K precursor molecules of IgE-potentiating factor. Human IgE-potentiating factors were obtained from a human T-T hybridoma. The factors have affinity not only for human IgE but also for rat IgE and selectively enhanced antigen-induced IgE response of rat lymphocytes. The same hybridoma could be switched to form IgE-suppressive factor by the addition of glycosylation-inhibiting factor (GIF) during its biosynthesis. Purified GIF has immunosuppressive activity in the mouse. This factor suppressed the primary IgE and IgG antibody responses in the mouse, and markedly suppressed ongoing IgE antibody formation.

Unique features of human basophilic granulocytes developed in in vitro culture.

Human basophilic granulocytes were obtained in suspension culture of mononuclear cells from umbilical cord blood. Precursors of basophils in the cord blood are nonadherent cells and bear neither surface Ig nor T cell marker. Cultured basophils contain chondroitin-4-sulfate proteoglycan, 1.74 micrograms histamine per 10(6) cells in average, and bear 120,000-380,000 IgE receptors per cell. The IgE receptor molecule has a molecular weight of approximately 64,000 daltons. Human, rat and mouse IgE bind to the receptors with comparable high affinity and passively sensitize the cells for mediator release. Challenge of sensitized basophils with anti-IgE resulted in stimulation of phospholipid methylation, 45Ca uptake, release of both histamine and free arachidonic acid, and enhancement of phosphatidylinositol (PI) turnover. Evidence was obtained that the activation of membrane-associated proteolytic enzyme and methyltransferases is involved in subsequent IgE-mediated biochemical cascades such as PI turnover, Ca2+ uptake and mediator release.

Suppression of IgE synthesis in mouse plasma cells and B cells by rat IgE-suppressive factor.

Rat IgE-binding factors were assessed for the ability to regulate the IgE response of mouse spleen cells. A rat-mouse T cell hybridoma, 23B6, produced two species, i.e., 15,000 (15K) daltons and 30K daltons, of rat IgE-binding factors upon incubation with rat IgE. The 15K IgE-binding factor selectively suppressed the in vitro IgE response of DNP-KLH-primed BALB/c spleen cells to homologous antigen without affecting the IgG1 response, whereas the 30K IgE-binding factor failed to affect both the IgE and IgG1 responses. Incubation of IgE-producing hybridoma cells with the 15K IgE-binding factor resulted in a parallel decrease in the proportion of IgE-secreting cells, cytoplasmic IgE-containing cells, and sIgE+ cells. The same IgE-binding factor suppressed IgE formation by mouse plasma cells in DNP-KLH-primed or KLH-primed BDF1 mice without affecting the IgG1-forming cells. In contrast, the 30K IgE-binding factor failed to affect the formation of IgE by the hybridoma and the plasma cells. It was also found that incubation of the splenic lymphocytes with the 15K IgE-binding factor resulted in a decrease in sIgE+ B cells; the 30K IgE-binding factor failed to do so. The results indicate that the binding of the 15K IgE-binding factor to plasma cells and to sIgE+ B cells results in suppression of IgE synthesis in these cells.