Allergen-specific IgE production of committed B cells from allergic patients in vitro.

The allergen-specific in vitro IgE synthesis in blood leukocytes from patients with allergy was monitored outside the pollen season with recombinant and natural pollen allergens and was compared with the total IgE production. The addition of interleukin-4 (IL-4) and antibody to CD40 increased the amount of total IgE by up to 20-fold in the culture supernatants of peripheral blood leukocytes from patients with allergy that could be antagonized by a neutralizing anti-IL-4 antibody in a dose-dependent manner. In contrast to total IgE, the amount of allergen-specific IgE was not affected by IL-4, and anti-CD40 or anti-IL-4, treatment. With oligonucleotides specific for IgE, complementary DNA from the amino terminal of the IgE heavy chain could be reversely transcribed and amplified by polymerase chain reaction from RNA of patients' unstimulated blood leukocytes, indicating that the IgE secretion in the cultures is due to a de novo IgE synthesis. It is concluded that the peripheral blood of patients with allergy contains long-lived allergen-specific B cells, which are not responsive to IL-4-mediated signals. These results may have implications for attempts to modulate specific IgE production in allergic patients with cytokines or cytokine antagonists.

Immunoglobulin E plus antigen challenge induces a novel intercrine/chemokine in mouse mast cells.

In an attempt to characterize genes participating in the allergic late phase reaction, we have isolated a novel intercrine/chemokine (called MARC) from a cDNA library of the stimulated mouse mast cell line, CPII. As measured by Northern blotting, it is strongly upregulated at the mRNA level after the physiological challenge of the cells with immunoglobulin (Ig)E plus antigen. Unstimulated cells completely lack significant, stable expression, as do a number of other, different cell lines (uninduced and induced) and mouse tissues. In contrast to the Northern blot analysis, a polymerase chain reaction (PCR) analysis, performed on CPII cells and on Percoll gradient purified mouse peritoneal mast cells, revealed a basal level of transcription in the uninduced stage. After 2 h of IgE plus antigen challenge, a quantitative reverse transcriptase-PCR, using a spiked in MIMIC, showed a level of transcripts more than 100-fold higher in the CPII cells and 5-20-fold higher in purified mouse peritoneal cavity mast cells. This rapid induction after the Fc epsilon RI challenge, the identification of the gene as a member of the chemokine family, and its upregulated expression in peritoneal mast cells, all suggest an involvement in certain acute and chronic pathological mast cell-driven diseases.

Immune response to tumor antigens in a patient with colorectal cancer after immunization with anti-idiotype antibody.

An active vaccination protocol was performed on one patient with colon carcinoma as a pilot to a prospective randomized double-blind clinical trial with the vaccine SDZ SCV 106. This vaccine is an anti-idiotype goat antibody to the monoclonal antibody 17-1A, which is directed against the tumor antigen 17-1A. To study the effect of the therapy on the immune reactivity, several tests were performed to detect anti-tumor antibodies in the serum as well as in eluates of metastatic tissue. Furthermore metastases removed from the lung were examined by immunohistochemistry. The results suggest that the humoral and cellular immune reactivity against the tumor are enhanced.

Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family.

Human glioblastoma cells secrete a peptide, termed glioblastoma-derived T cell suppressor factor (G-TsF), which has suppressive effects on interleukin-2-dependent T cell growth. As shown here, complementary DNA for G-TsF reveals that G-TsF shares 71% amino acid homology with transforming growth factor-beta (TGF-beta). In analogy to TGF-beta it is apparently synthesized as the carboxy-terminal end of a precursor polypeptide which undergoes proteolytic cleavage to yield the 112 amino-acid-long mature form of G-TsF. Comparison of the amino-terminal sequence of G-TsF with that of porcine TGF-beta 2 and bovine cartilage-inducing factor B shows complete homology, which indicates that we have cloned the human analogue of these factors. It is tempting to consider a role for G-TsF in tumor growth where it may enhance tumor cell proliferation in an autocrine way and/or reduce immunosurveillance of tumor development.

Preliminary studies on the inflammatory stimulus induced proteins in mouse resident peritoneal macrophages.

Chemotaxis, release of lysosomal enzymes, synthesis of eicosanoids and phagocytosis are some of the important functions mediated by macrophages. Active protein synthesis have been implicated as an essential step in the mediation of these vital physiological functions. The proteins synthesized during the inflammation with microbial agents have not been identified. In the current investigation, we report the synthesis of five proteins of molecular weights 72,000; 70,000; 40,000; 34,000; and 32,000 in mouse peritoneal macrophages after incubation with inflammatory stimuli derived from microorganisms. A possible role for these proteins in phagocytosis was suggested.

T cell suppressor factor from human glioblastoma cells is a 12.5-kd protein closely related to transforming growth factor-beta.

T cell suppressor factor produced by human glioblastoma cells inhibits T cell proliferation in vitro and more specifically interferes with interleukin-2 (IL-2)-dependent T cell growth. Here we report the purification of this factor from conditioned medium of the human glioblastoma cell line 308. Amino-terminal sequence analysis of the 12.5-kd protein demonstrates that eight out of the first 20 amino acids are identical to human transforming growth factor-beta. Purified glioblastoma-derived T cell suppressor factor and transforming growth factor-beta from porcine platelets inhibit both IL-2-induced proliferation of ovalbumin-specific T helper cells and lectin-induced thymocyte proliferation with similar specific activities. If released by glioblastoma cells in vivo, the factor may contribute to impaired immunosurveillance and to the cellular immunodeficiency state detected in the patients.

Effect of cyclosporin A on immunity to Listeria monocytogenes.

The effect of the immunosuppressive drug cyclosporin A (CS-A) on immunity to the facultative intracellular bacterium Listeria monocytogenes was investigated in unprimed and primed mice. Different treatment protocols were followed to evaluate the time dependence of CS-A-mediated immune suppression and the effect of CS-A on immunological memory to L. monocytogenes. The effect of CS-A was observed only during and after activation of T cell-mediated immunity, whereas early resistance exerted by macrophages assessed 6 and 70 min after challenge remained unaffected. CS-A suppressed efficient elimination of L. monocytogenes even when given after day 3 of a primary infection. This contrasts with findings in other models, including viral infections, where CS-A must be administered very early in an immune response to suppress it. CS-A suppressed antibacterial resistance in mice primed at various times before challenge; suppression of protection was time dependent and was virtually complete in livers, whereas CS-A-resistant memory persisted in spleens for up to 10 months.

Recombinant interleukin 2 enhances spontaneous insulin-dependent diabetes in BB rats.

Treatment of BB rats with recombinant interleukin 2 (IL2) enhanced the development of spontaneous diabetes in these animals. A dose of 20 micrograms IL 2/kg body weight was administered twice daily for 80 days starting at 42 days of age. The rate of diabetes was doubled after IL 2 administration (53% vs. 23%) and the onset of diabetes was found to be accelerated by a mean of 18 days. Histological analysis showed enhanced inflammation of islets and in addition interstitial pancreatis. It is concluded that IL 2 has a regulatory effect on spontaneous organ-specific autoimmunity.

Early events during primary activation of T cells: antigen receptor cross-linking and interleukin 1 initiate proliferative response of human T cells.

To study early events during primary activation of human T cells, a simple method was developed which simultaneously allows positive selection of T cells from peripheral blood lymphocytes (PBL) and their polyclonal, antigen receptor-mediated stimulation with anti-T3 monoclonal antibodies. In the absence of accessory cells, T cells activated with matrix-bound OKT3 express high levels of the Tac antigen within 15 h and produce interleukin 2 (IL2). Tac expression was further enhanced by addition of exogenous IL2. However, under these conditions purified T cells were unable to mount a proliferative response, whereas unfractionated PBL proliferated already after 24 h of culture. This unresponsiveness of purified T cells could be overcome by either re-addition of low numbers of autologous accessory cells or semipurified human IL1. As IL1 had no significant effect on Tac expression of T3-stimulated T cells, we conclude from these data that IL1 exerts in addition to its influence on IL2 production an effect, which allows antigen receptor-triggered T cells to enter the cell cycle.

Lipopolysaccharide significantly enhances erythrophagocytosis but marginally stimulates the phagocytosis of Staphylococcus aureus in mouse peritoneal macrophages.

The influence of lipopolysaccharide (LPS) on phagocytic and bactericidal functions of normal mouse peritoneal macrophages was investigated. Preincubation of macrophages with LPS enhanced their capacity for phagocytosis of antibody coated sheep red blood cells 5-fold, but phagocytosis of antibody coated Staphylococcus aureus was enhanced only 1.2-ld. Phagocytosis and intracellular killing of unopsonised or normal rabbit serum opsonised S. aureus was not affected by the LPS treatment of macrophages.

Preliminary studies on the induction of a 120 000 molecular weight protein in resident peritoneal macrophages by arachidonic acid.

Exogenous arachidonic acid induced the synthesis of a 120 000 molecular weight protein in resident peritoneal macrophages. The induction of this protein is specific to the presence of arachidonic acid in the culture medium and is not induced by the presence of other fatty acids, irrespective of their chain length or degree of unsaturation. The protein induced is not a secretory protein and is not formed as a result of the processing of preexisting proteins in macrophages. In addition to arachidonic acid, prostaglandin E2 also induced the synthesis of 120 000 molecular weight protein in macrophages.

Incorporation of palmitic acid or oleic acid into macrophage membrane lipids exerts differential effects on the function of normal mouse peritoneal macrophages.

Normal mouse peritoneal macrophages were enriched with either palmitic acid (16:0) or oleic acid (18:1). Normal or oleic acid-enriched macrophages showed 3-4-fold greater erythrophagocytic capacity as compared to palmitic acid-enriched macrophages. Staphylococcus aureus uptake was only moderately decreased in palmitic acid-enriched macrophages. Fatty acid modifications did not influence the ability of macrophages to kill intracellular bacteria or to generate superoxide anions after stimulation with phorbol myristate acetate or opsonized zymosan.

Receptor remodeling and regulation in the action of epidermal growth factor.

Epidermal growth factor (EGF) initiates a wide variety of events when added to responsive cultured cells. These range from early events requiring only brief exposure to EGF, e.g., stimulation of transport of amino acids or ions, to later events such as commitment of cells to a round of DNA synthesis, a process requiring 6 h or more of continuous exposure to hormone. EGF binding is followed first by phosphorylation of EGF receptors, which can be detected in purified membranes and permeabilized cells, and then by internalization and proteolytic processing of receptors in lysosomes. Native 160,000-dalton EGF receptors contain a site that is not exposed on the cell surface and is highly sensitive to cleavage by an endogenous protease, which yields a 145,000-dalton receptor fragment that retains phosphate acceptor activity. Cleavage of receptor at a trypsin-sensitive site, also not exposed to the cell surface, yields a 115,000-dalton fragment that binds EGF, but contains no phosphorylated species. The data indicate that the phosphate acceptor sites on EGF receptors are localized on a 45,000-dalton cytosolic region.

Modulation of epidermal growth factor receptors on 3T3 cells by platelet-derived growth factor.

Platelet-derived growth factor does not compete with epidermal growth factor (EGF) for binding to EGF receptors on the murine 3T3 cell surface, but it modulates EGF receptors in two ways: (i) it induces a transient down regulation of EGF receptors and (ii) it inhibits EGF-induced down regulation of EGF receptors. These data suggest a common cellular internalization mechanism for the receptors for both hormones.

Effects of cytochalasin B and enucleation on EGF receptor down regulation.

The loss of epidermal growth factor (EGF) binding activity on cultured murine 3T3 cells exposed to EGF (EGF receptor down regulation) was determined in colchicine treated cells, cytochalasin B treated cells, and untreated cells. Neither colchicine nor cytochalasin B altered the affinity of the receptor for EGF, but colchicine decreased maximal EGF binding activity by 20%. The maximal extent of EGF receptor down regulation was similar in colchicine treated cells and cytochalasin B treated cells, but the rate of receptor down regulation was higher in cytochalasin B treated cells. Cytoplasts produced by subjecting cytochalasin B treated cells adhering to the substratum to centrifugal force responded to EGF with nearly normal down regulation kinetics. The results suggest that the cytoskeleton is not obligatorily involved in EGF-induced EGF receptor down regulation.

Molecular identification and properties of two cell surface receptors playing roles in mitogenesis and epigenesis.

Studies leading to the identification of cell surface receptor macromolecules which specifically recognize a hormone, epidermal growth factor (EGF), or gp70, the coat antigen of the C-type RNA tumor viruses, have been described. EGF receptors are internalized and processed by lysosomal protease action after interaction of receptor and EGF. Other hormones, which resemble EGF in their ability to trigger mitogenesis in cultured cells, interact with the EGF receptor in a yet to be defined, but probably indirect way, decreasing the number of EGF binding sites on the cell surface. The evidence at this point indicates that these hormones interact with EGF receptors through the communal utilization of a shared cellular mechanism for internalization of their receptors and the EGF receptors. The receptor for gp70 is not internalized in response to gp70 binding, but is instead shed into the medium by cultured cells. This receptor protein BPgp70 has now been isolated and purified to apparent homogeneity. Antibodies to BPgp70 completely block gp70 binding to cells at low concentration, indicating that BPgp70 is the physiological receptor for gp70.