Human peripheral blood basophils primed by interleukin 3 (IL-3) produce IL-4 in response to immunoglobulin E receptor stimulation.

In contrast to most cytokines, interleukin 4 (IL-4) expression is restricted to T lymphocytes, with the exception of mast cell lines and mast cells, as more recently demonstrated in rodents. Little is known, however, about the capacity of human nonlymphoid cells to produce IL-4. In this study we show that mature human basophils are capable of expressing IL-4 and examine the regulation of IL-4 production in comparison with the lipid mediator leukotriene C4. IL-4 was produced upon immunoglobulin E receptor (IgER) activation of basophils cultured with IL-3, a cytokine previously shown to prime these cells for enhanced release of inflammatory mediators. In some experiments, IL-3 or IgER activation alone also induced IL-4 production close to the detection limit. The effect of IL-3 on IgER-dependent IL-4 expression was dose and time dependent: maximal IL-4 production occurred between 18 and 48 h preexposure of basophils to 3-10 ng/ml IL-3. IgER-induced IL-4 synthesis and release by basophils cultured with IL-3 was rapid and complete after 6 h. In contrast to IL-3, other cytokines (IL-5, granulocyte/macrophage colony-stimulating factor, and nerve growth factor) that also prime basophils for enhanced histamine and leukotriene C4 release did not promote IgER-induced IL-4 synthesis. Basophils appear to secrete a "TH2-like" cytokine profile since no detectable IL-2 or interferon gamma was produced upon IgER activation. Mononuclear cells (depleted of basophils), cultured in parallel, did not release IL-4 in response to IL-3 and/or IgER activation, and produced approximately ten times less IL-4 than basophils upon nonspecific activation by phorbol ester and calcium ionophore. Thus, human basophils are an important cellular source of IL-4, and may, therefore, in addition to their inflammatory effector functions, also regulate the differentiation of T helper cells and B cells, in particular in allergic diseases.

Interferon alpha increases the frequency of interferon gamma-producing human CD4+ T cells.

An increased ratio of T helper type 2 (Th2)- vs Th1-like cells contributes to the immune dysregulation in allergic disease situations and in many chronic infections, including AIDS. Th2-type immune responses are characterized by Th cells that produce increased levels of interleukin-4 (IL-4) and decreased levels of interferon gamma (IFN-gamma). The induction of either a Th1- or a Th2-like phenotype may be critically controlled by the antigen-presenting cells and their cytokines, e.g., IFN-alpha. In this study we have determined the frequencies of potential IL-4- and/or IFN-gamma-producing T cells in the peripheral blood of randomly selected healthy individuals, and analyzed whether IFN-alpha controls IL-4 and/or IFN-gamma production. Purified CD4+ or CD8+ T cells were stimulated for 24 h via the T cell receptor/CD3 complex in the presence or absence of IFN-alpha, and single IL-4- and IFN-gamma-secreting cells were detected in enzyme-linked immunospot assays. In the absence of IFN-alpha, CD4 cells produced IFN-gamma at frequencies of 1:50-300, and produced IL-4 at frequencies of 1:110-<1:100,000. Addition of IFN-alpha during the activation of CD4 cells increased the levels of IFN-gamma mRNA. As a consequence, the numbers of IFN-gamma-producing CD4 cells and the amounts of secreted IFN-gamma increased 10-fold. In contrast, IFN-alpha did not increase the frequency of IL-4-secreting CD4 cells. In the absence of IFN-alpha, addition of exogenous IL-4 to cultures of CD4 cells suppressed IFN-gamma secretion by 70%. However, in the presence of IFN-alpha, IL-4 did not display any suppressive effect. Compared with CD4 cells, CD8 cells produced IFN-gamma more frequently (1:5-10) but IL-4 less frequently (1:5,300 to < 1:100,000). IFN-alpha did not display any effect on the frequency of either IFN-gamma or IL-4 production by CD8 cells. Taken together the results indicate that IFN-alpha increases the frequency of IFN-gamma-secreting CD4 Th cells and antagonizes the suppressive effect of IL-4 on IFN-gamma production. As a consequence, IFN-alpha may favor the induction and maintenance of Th1-like cells and thereby counteract Th2-driven allergic immune responses.

Receptors for IgG: subclass specificity of receptors on different mouse cell types and the definition of two distinct receptors on a macrophage cell line.

To evaluate subclass specificity and aggregate size requirements of IgG receptors on mouse cells, we measured binding of radiolabeled monomeric and BDB-aggregated mouse myeloma proteins fractionated into various sizes by means of gel filtration. Monomers, tetramers, and high molecular weight (approximately 10(7) daltons) aggregates were used. The various cells and cell lines studied could be segregated into three patterns of reactivity: (a) Macrophage and macrophage-like cell lines bound monomer IgG2a preferentially; high molecular weight IgG aggregates bound as follows: IgG1 = IgG2b = IgG2a. (b) Lymphoid lines D2N and S49 showed no capacity to bind monomer IgG2a; high molecular weight aggregates bound as follows: IgG1 = IgG2b less than IgG2a. (c) Other Thy-1-positive lymphoid cell lines (EL4 and L5178) and normal T and B cells showed no capacity to bind monomer IgG; high molecular weight IgG aggregates bound to a lesser extent than to cells of the first two categories in the following manner: IgG1 less than IgG2b greater than or equal to IgG2a. The variable pattern of reactivity of the macrophage-like cell lines with monomer and aggregated IgG suggested that two distinct receptors for IgG were present: one capable of binding IgG2a and another capable of binding all aggregates. Further evidence for this hypothesis was obtained by analysis of the inhibitory capacity of different IgG subclasses on the binding of aggregated IgG and monomer IgG2a to P388 cells. Inhibition of monomer IgG2a binding was effected only by monomer or aggregated IgG2a, whereas inhibition of binding of aggregated IgG1 or IgG2b was noted with aggregates of all three subclasses with some preferential inhibition by monomer IgG2b being observed. Furthermore, monomer IgG2b binding was preferentially inhibitable by monomer IgG2b. It is postulated from these data that two receptor sites are present on this macrophage-like cell line, one reactive with aggregates of all three subclasses as well as monomer IgG2b, and another receptor specific for monomer IgG2a which also binds aggregated IgG2a. Support of this concept was obtained by trypsinization experiments in which the binding of monomer IgG2a was markedly decreased by trypsin treatment of cells, whereas the binding of aggregated IgG2b was unaffected by this treatment.

Interleukin 4 is localized to and released by human mast cells.

Recent attention has focused on the T helper type 2 (Th2) lymphocyte as a source of interleukin 4 (IL-4) in allergic disease. However, Th2 cells themselves require a pulse of IL-4 to initiate this synthesis. Here we provide immunohistochemical evidence of IL-4 localization to human mast cells of the skin and respiratory tract, and demonstrate that immunoglobulin E-dependent stimulation of purified human lung mast cells leads to the rapid release of IL-4 into the extracellular environment. We propose that mast cell activation in an allergic response provides a rapid and local pulse of IL-4 into the local environment essential for the triggering of T lymphocytes into sustained IL-4 production and to initiate inflammatory cell accumulation and activation.

Specific V gene usage in anti-phosphorylcholine IgE antibodies.

Three hybridomas from phosphorylcholine(PC)-KLH immunized BALB/c mice producing IgE antibodies against the PC hapten were investigated for their fine specificity to the hapten and usage of V gene segments in H- and L-chains. All three IgE antibodies recognize the entire azophenyl-PC hapten. They are T15 Id negative and do not bind to the natural PC determinant expressed by the Streptococcus carbohydrate R36A. T15 Id positive IgE antibodies could neither be elicited by immunization in detectable amounts nor generated by the cell fusion technique. By using the Southern blot technique and nucleotide sequence analysis of PCR amplified VHDJH and VLJL rearrangements, we have demonstrated that the three IgE anti-PC hybridomas use the VH1-DSP2-JH2, the VHOX1-DSP2-JH3 or the VH36-60-D-JH2 gene segment combinations for the H chain together with the V kappa 1C-J kappa 1, V kappa 1C-J kappa 2 or V lambda 1-J lambda 1 genes for the L chains. Except for the VH36-60, the same gene segments were found in different combinations in anti-PC antibodies of other Ig classes than IgE. However, high rates of somatic mutations are expressed in both VH1 of the H chain and in V kappa 1C of the L chain. The VH36-60 is expressed in antibodies with the major Id of the azophenyl-arsonate (Ars) response and VHOX1 generally contributes to the phenyl-oxazolone specificity. This suggests that these V genes are involved in the recognition of the azophenyl moiety of the coupled PC hapten. Thus PC-KLH specific IgE antibodies utilize mutated VH1 and/or VH/VL gene segment combinations which are involved in binding of the azophenyl spacer. These IgE are therefore specific for azophenyl-phosphorylcholine, unlike antibodies normally expressed against the Streptococcus PC determinant in mice. The genetic diversity and the high mutation rates indicate that the specific B cells develop later in the immune response. Thus, they represent newly generated specificities of so-called group II anti-PC antibodies and are not isotype-switch descendants from already existing T15 Id positive IgM antibodies.

T cell dependent differentiation of human B cells: direct switch from IgM to IgE, and sequential switch from IgM via IgG to IgA production.

Ig production by splenic human B cells that express different surface Ig isotypes were analysed in limiting dilution cultures. Therefore, FACS sorted IgM+, IgG+ and IgA1+ B cells were stimulated with PMA-activated EL4 thymoma cells as helper cells in the presence of IL-2 and IL-4. We found that at least every second B cell responded in vitro and secreted the antibody corresponding to its surface Ig isotype. IgE secreting cells developed from surface IgM+ D+ cells (1/31 to 1/167), but not from IgG+ or IgA1+ cells (much less than 1/5000). Negative signalling of the IgM+ B cells by addition of anti-IgM antibodies into the cultures reduced the number of single IgM producing cells by greater than 85%, and completely inhibited IgE switch. In contrast, anti-IgG and anti-IgA antibodies did not reduce the IgE response. The results indicate a direct switch from IgM to IgE secretion in vitro. In contrast to IgE, IgA secreting cells developed from IgM+D+ (1/30 to 1/51) and from IgG+ B cells (1/14 to 1/25). Negative signalling of the IgG+ B cell subset within total B cells by anti-IgG antibodies suppressed the development of IgG as well as IgA producing cells, but did not inhibit IgM and IgE responses. This indicates a sequential switch from IgM via IgG to IgA. Taken together, this study indicates that IgE secreting cells are derived directly from IgM+D+ B cells by non-sequential switching, whereas IgA producing cells preferentially develop by sequential switching via IgG+ B cells.

Fine specificity and heavy chain V gene usage of antibodies to the phosphorylcholine hapten.

In the course of the immune response to PC-KLH in mice, two major groups of anti-PC antibodies are expressed which differ in fine specificity, Id expression and isotype preference. Group I antibodies react predominantly with the PC moiety of the hapten and display mostly the T15 Id. On the other hand, Group II antibodies recognize the PC hapten including the azophenyl spacer to the carrier and are T15 Id-. In order to investigate the relationship between the two antibody populations, we analyzed the composition of 20 anti-PC hybridomas of all isotypes with respect to their fine specificity and VH gene segment utilization. Characterization of the antibodies by hapten inhibition analyses with PC and p-nitro-phenyl-PC indicated that eight hybridomas secreted Group I and 12 Group II antibodies. Southern blot analysis of total genomic DNA using a germ-line JH probe demonstrated that, with the exception of aPC-111-1, all Group II cells utilized gene segments distinct from those of Group I. In contrast to the restricted VH gene usage in Group I hybridomas, a more pronounced heterogeneity in H chain V gene rearrangements in Group II cells was observed. We found three groups of hybridomas with identical VH gene rearrangements. Two hybridomas used a VH gene segment of the 7183 VH gene family. Three lambda 1 bearing Group II hybridomas shared a common hybridizing band. One of these, aPC-56-1, is known to utilize the same VH gene segment as MOPC 141. Finally, three cell lines, one of which (aPC-104-8) utilizes the VH1-DH-JH2 rearrangement, showed fragments of identical sizes. These results emphasize the independent origin of Group I and Group II anti-PC antibodies and demonstrate a larger germ-line repertoire of Group II antibodies as well as a less restricted use of particular VH genes relative to that of Group I.

Mechanism of inhibition of human renin by monoclonal antibodies.

The mechanism by which monoclonal antibodies directed against human renin (R3-36-16 and R3-47-10) inhibit renin activity was investigated using various substrates. Both antibodies acted as potent inhibitors of human renin activity when human angiotensinogen was used as a substrate. However, their effects differed clearly in the presence of synthetic tetradecapeptide. When low concentrations of tetradecapeptide were used as substrate, renin activity was only partially inhibited by R3-47-10, whereas it was stimulated by R3-36-16. At higher synthetic substrate concentrations, both antibodies stimulated angiotensin I production. This effect was independent of the pH. Both antibodies exerted their effects in the presence of CGP 29287, a peptidic transition-state competitive renin inhibitor, indicating that their binding sites differed from that of CGP 29287. In combination, the stimulatory effect of R3-36-16 was not blocked by R3-47-10, but the inhibition produced by R3-47-10 was reversed by R3-36-16. Both antibodies may prevent the large natural substrate angiotensinogen from entering the enzymatic cleft by steric hindrance. At a low substrate concentration, R3-47-10 may also partially hinder the access of synthetic tetradecapeptide into the active cleft by steric hindrance. In contrast, the stimulating effect of both antibodies may be due to a conformational change in the renin molecule, allowing an increased access of tetradecapeptide or a more rapid release of the product from the enzymatic cleft.

Characterization of a monoclonal antibody specific for human active renin.

We have identified and characterized an anti-human renin monoclonal antibody R1-20-5 that is selective for human active renin. R1-20-5 binds active renin with a dissociation constant (Kd) of 2.5 x 10(-7) M/l and inhibits renin enzymatic activity with an inhibitory constant (IC50) of 1.4 x 10(-8) M/l. R1-20-5 competes with a synthetic renin inhibitor for binding with renin, demonstrating further that it is binding to or close to the active site. This antibody does not bind prorenin in human plasma or recombinant prorenin expressed by L-929 fibroblasts transfected with human renin gene. Furthermore, trypsin activation of prorenin resulted in immunoreactivity of the activated prorenin toward the antibody. In addition, an immunoaffinity column of R1-20-5 coupled to Sepharose retained active renin but had a low affinity for prorenin. A sensitive and rapid solid phase radioimmunoassay for active renin was developed using a "sandwich" technique employing R1-20-5 and a second non-active site-directed monoclonal antibody to human renin. Renin levels in human plasma samples were determined by the standard enzymatic assay, and by the direct radioimmunoassay for active renin, before and after trypsin activation. Trypsin treatment of plasma resulted in parallel increases in both the plasma renin enzymatic activity and in the plasma active renin concentration as measured by the direct radioimmunoassay. Overall, plasma immunoreactive active renin concentration correlated significantly with plasma renin enzymatic activity (r = 0.96, p less than 0.001). In summary, the monoclonal antibody R1-20-5 is selective for human active renin and should be a very useful tool for studies of the active enzyme in humans.

Methods for binding cells to plastic: application to a solid-phase radioimmunoassay for cell-surface antigens.

Two methods are described for attaching cells to plastic plates such that they may be used for antibody binding assays. In the first method, lymphoid cells or erythrocytes were attached to the wells of plastic plates using glutaraldehyde. This resulted in monolayers of fixed cells which retained surface antigens and were stable to storage. The second method involved binding of unfixed cells to the plastic surface by means of antibodies non-specifically adsorbed to the plate. Both methods resulted in cell layers which remained attached to the plate during the washing and incubation procedures of a radioimmunoassay. The cell layers were shown to be suitable for screening the product of hybrid cell lines for the presence of monoclonal antibodies to cell-surface antigens.

Production and characterization of mouse antibodies against the brain lipid sulfatide.

One of the lipids in the myelin sheet is sulfatide, a galactosphingolipid. Our aim was to develop a test system to detect and characterize antibody against this lipid. Mouse anti-sulfatide antibody was estimated by a solid-phase microtube assay with labeled Staphylococcus aureus protein A. With labeled rabbit anti-mouse Ig antibody, there was no difference between mouse anti-sulfatide serum and control sera from animals immunized with unrelated antigens. Results show that BALB/c mice produce antibodies against sulfatide if this low molecular weight compound is injected together with cholesterol, lecithin and bovine serum albumin. The antisera are specific for sulfatide but crossreact with galactocerebroside. However, mouse IgM antibody binds to sulfatide-coated polyvinyl plates non-specifically. Thus, only test procedures which avoid detection of IgM antibody can be used to estimate antibody specific for sulfatide and probably also for other lipophilic compounds.

Detection of and discrimination between total and free human interleukin-4 and free soluble interleukin-4 receptor by ELISA.

Interleukin-4 (IL-4) signaling is initiated by binding of IL-4 to the high-affinity IL-4 receptor alpha-chain and subsequent interaction with the common gamma-chain. Soluble forms of the extracellular domain of the alpha-chain (sIL-4R) were shown to be present in biological fluids and, dependent on the concentration, enhance or inhibit IL-4 activity by forming IL-4/sIL-4R complexes. To discriminate between free and potentially active IL-4 from the inactive and complexed form, we have established a set of new ELISA systems for the measurement of human IL-4 in its distinct forms. To select suitable pairs of anti-IL-4 antibodies, a chequerboard interference analysis with six highly-selective human IL-4 specific monoclonal antibodies was performed. For the determination of total IL-4, a monoclonal capture antibody was used that binds IL-4 outside the binding site of the IL-4R alpha-chain. Another antibody recognizing an epitope of the alpha-chain binding site was chosen for the detection of free IL-4. The binding of this antibody was inhibited in a dose-dependent fashion by recombinant sIL-4R. Assays for both total and free IL-4 exhibited a sensitivity of 8 pg/ml and a dynamic range up to 1000 pg/ml. Human sIL-4R was detected by two monoclonal antibodies directed against different epitopes. This ELISA was inhibited by recombinant IL-4 suggesting the measurement of predominantly free sIL-4R. Complexes between soluble IL-4R and IL-4 were detected by a monoclonal anti-sIL-4R antibody in combination with an anti-IL-4 antibody. When supernatants of activated T cells were analyzed, the majority of the IL-4 was in free form. The amount of complexed IL-4 was low as indicated by the fact that most of total IL-4 could be detected as free IL-4. Although values obtained for complexed IL-4 correlated with the difference between total and free IL-4, precise values could not be determined, presumably due to the dynamic nature of the complex between the two proteins. We suggest that the ability to quantitate total and free IL-4 in combination with sIL-4R may provide a new insight of the role that IL-4 plays in different pathophysiological conditions.

Non-specific binding of mouse IgM antibodies to lipid antigens.

Galactocerebroside (GC) is a major component expressed on the surface of oligodendrocytes and Schwann cells. It plays a role in neuronal-glial interaction that results in myelinization. In this paper we describe a method to estimate anti-GC antibodies by ELISA. Furthermore we found that IgM antibodies of mice immunized with GC-unrelated antigens, and which are not specific for GC, bind in a non-specific way to this compound. Thus only assays which avoid the detection of IgM antibody can be used to estimate anti-GC antibodies. Moreover, we showed that serum of immunized mice bind also non-specifically to brain cells of newborn mice.

The IgE antibody response to the phosphorylcholine hapten.

The immune response to the phosphorylcholine (PC) hapten elicited by PC-keyhole limpet hemocyanin (KLH) is composed of 2 groups of antibodies with specificity to either PC or phenylphosphorylcholine which were designated as group I and II anti-PC antibodies, respectively. We demonstrate that anti-PC IgE antibody expression is restricted to group II antibodies and does not display the T15 idiotype. Accordingly anti-PC IgE antibodies recognize the same epitope (PC-phenyl) as IgG1, IgG2a and IgG2b antibodies which is different from that (PC) recognized by IgM and IgG3 antibodies. A monoclonal anti-PC IgE antibody, representing group II characteristics was established. From amino acid sequences of light chains of purified group I and II antibodies from serum as well as of monoclonal representatives thereof it appears that both populations are relatively homogenous and represent independent clonal expressions. Nevertheless the formation of anti-PC IgE antibodies in mice can be suppressed by isologous anti-T15 anti-idiotypic antiserum. This antiserum, however, crossreacts with different anti-PC antibodies including monoclonal group II anti-PC IgE antibodies and is composed of a large number of anti-idiotopes. An analyses performed with monoclonal anti-T15 idiotopes demonstrates that some but not all antibodies suppress the formation of anti-PC IgE. We conclude that syngeneically induced anti-idiotypic interactions may display regulation of a wide range of specificities affecting responses to various antigenic determinants.

Radioimmuno positron emission tomography with monoclonal antibodies: a new approach to quantifying in vivo tumour concentration and biodistribution for radioimmunotherapy.

Radioimmunodetection of tumours with monoclonal antibodies is becoming an established procedure. Positron emission tomography (PET) shows better resolution than normal gamma camera single photon emission tomography and can provide more precise quantitative data. Thus, in the present study, these powerful methods have been combined to perform radioimmuno PET (RI-PET). Monoclonal antibodies directed against carcinoembryonic antigen (CEA) an IgG, its F(ab')2 and a mouse-human chimeric IgG derived from it were labelled with 124I, a positron-emitting radionuclide with a convenient physical half-life of four days. Mice, xenografted with a CEA-producing human colon carcinoma, were injected with the 124I-MAb and the tumours were visualized using PET. The concentrations of 124I in tumour and normal tissue were determined by both PET and direct radioactivity counting of the dissected animals, with very good agreement. To allow PET quantification, a procedure was established to account for the presence of radioactivity during the absorption correction measurement (transmission scan). Comparison of PET and tissue counting indicates that this novel combination of radioimmunolocalization and PET (RI-PET) will provide, in addition to more precise diagnosis, more accurate radiation dosimetry for radioimmunotherapy.

[Immune response and pathophysiology of the allergic reaction]. / Immunantwort und Pathophysiologie der allergischen Reaktion.

The allergic immune response is characterized by a number of cellular and molecular interactions. Allergens are taken up through the respiratory or digestion tract or the skin by dendritic cells, B cells or macrophages. After phagocytoses and processing, fragments of allergens are presented to the allergen-specific T cells. By this process, allergen-reactive T cells are induced, which are predominantly of the Th2 type and which secrete the cytokines IL-4, IL-5 and IL-10. In contrast, during a normal immune response to bacterial or viral allergens, T cells of the Th1 type are induced, which produce IFN gamma and IL-2 but not Th2 cytokines. A direct contact of Th2 cells with B cells results in activation of B cells. The Th2 cytokine IL-4 instructs B cells to switch from IgM to IgE antibody production. IgE antibodies play a central role in the induction of allergic diseases. IgE antibodies are taken up by basophils and mast cells by virtue of high-affinity receptors for IgE on these cells. Allergen confrontation leads to the activation of such IgE-sensitized cells, which results in the release of various mediators such as histamine, leukotrienes and prostaglandins; together, they induce the clinical manifestations of allergic reactions. Recent findings have shown that mast cells (and basophils) from atopic tissue are able to produce cytokines such as IL-4 and can thereby induce IgE antibody production. IL-5 generated by allergen-reactive Th2 cells attracts and activates eosinophils, which are responsible for tissue destruction in allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

T15 dominance in BALB/c mice is not controlled by environmental factors.

The effects of the environment on the expression of T15 in the in vivo anti-PC response of BALB/c mice were analyzed. T15 dominance in young BALB/c mice was independent of the expression of T15 dominance in either parent, because the offspring of parental mice that were suppressed for T15 production presented antibody responses dominated by the T15 idiotype. Also, dominant T15 expression was independent of living microorganisms; mice raised in conventional, specific pathogen-free or germfree conditions mounted similar T15 dominant antibody responses. Furthermore, T15 expression was independent of the conventional diet, because mice raised on a synthetic diet produced T15-dominant antibody responses. Moreover, mice that received a synthetic diet under germfree conditions also produced T15 dominant antibody responses. Thus, the generation of T15 dominance in BALB/c mice appears to be independent of environmental factors and within the context of the present and earlier results, originates at the level of B cell-mediated clonal selection/regulation, genetic mechanisms concerning Ig gene rearrangement and expression and/or the fine specificity of the combining site for antigen on the B cell.

Autoreactive or self restricted?

Putative major histocompatibility complex (MHC) class II-specific T cell clones were isolated from nominal antigen-primed T cell populations propagated in vitro under antigen selection. However, unlike the proliferative responses of nominal antigen-specific, MHC class II-restricted T cell clones derived from the same population, the former's MHC-specific proliferative responses required that the culture medium be supplemented with serum. The role of xenogeneic antigens in the generation of this category of "autoreactive" T cells is discussed.