Analysis of murine soluble Fc epsilon RII sites of cleavage and requirements for dual-affinity interaction with IgE.

The low-affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal sequence homologous to C-type animal lectins. Between this region and the membrane is a repetitive sequence predicted to form an alpha-helical coiled-coil and is termed the stalk region. The Fc epsilon RII is proteolytically cleaved when at the cell surface in this stalk region. Both the 38 Kd and 28 Kd major released fragments were isolated from culture media and N-terminal sequencing demonstrated that the cleavage sites were in the third and fourth repeat domains, respectively. The identified sites show no apparent similarity with the cleavage sites previously identified in human Fc epsilon RII. Recent studies have demonstrated that the intact Fc epsilon RII interacts with IgE with a dual-affinity, resulting from a multivalent interaction with the IgE Fc region; mutant Fc epsilon RII that have a disruption of the alpha-helical coiled-coil have a single low-affinity interaction consistent with a monomeric interaction with IgE. The soluble Fc epsilon RII were shown to interact with IgE with an affinity similar to these mutant Fc epsilon RII. Preparation of a chimeric Fc epsilon RII in which the transmembrane and cytoplasmic regions were replaced with sequences from Ly-49 revealed that these regions played no role in the multimeric association of the Fc epsilon RII necessary for dual-affinity interaction with IgE. In addition, a full-sized soluble Fc epsilon RII construct was expressed, and this molecule demonstrated increased capacity to interact with IgE.

Homotypic aggregation of murine B lymphocytes is independent of CD23.

CD23 is a low-affinity receptor for IgE (Fc epsilon RII). Functions attributed to CD23 not involving IgE suggest that it interacts with ligands other than IgE. CD21 has recently been described as a counter ligand for CD23. A number of lines of evidence have implicated CD23 as an adhesion molecule in human B cells. We have investigated the role of CD23 in homotypic B cell aggregation in the mouse, using lipopolysaccharide plus interleukin-4-induced aggregation as a model system. In this system high levels of aggregation are accompanied by a massive up-regulation of CD23 expression. However, in contrast to what has been observed in human B cells, we find no evidence of a role for CD23 in homotypic adhesion of murine B cells.

The oligomeric nature of the murine Fc epsilon RII/CD23. Implications for function.

The low affinity receptor for IgE (Fc epsilon RII/CD23) is a type II integral membrane protein with an extracellular C-terminal region homologous to C-type animal lectins. Immediately adjacent to this lectin homology region is a sequence that is predicted to form an alpha-helical coiled-coil stalk leading to dimer or trimer formation. This provides an explanation for the known self-associative capacity for the Fc epsilon RII. In this study the self-association to a trimer or tetramer is shown with rFc epsilon RII by chemical cross-linking and affinity purification on IgE columns. The data indicate that only the oligomeric form of Fc epsilon RII has sufficient affinity/avidity to bind to an IgE adsorbent. In contrast, Fc epsilon RII that is purified using anti-Fc epsilon RII mAb adsorbents has largely lost its capacity to bind IgE, as well as its capacity to self-associate, indicating that IgE recognizes the oligomeric form of the Fc epsilon RII. This phenomenon was further examined by performing detailed binding analysis of the mouse IgE/Fc epsilon RII interaction. A biphasic binding curve with high (2-7 x 10(7) M-1) and low (2-7 x 10(6) M-1) affinity binding was seen. Fc epsilon RII mutants were prepared that lack one or more of the 21 amino acid homologous repeat domains in the stalk region of the molecule. These mutant Fc epsilon RII molecules bound IgE with only a single low affinity (5-10 x 10(6) M-1). In addition, cross-linking analysis of one of these mutants demonstrated that it does not exhibit the receptor self-association seen for the intact Fc epsilon RII. Two chimeric Fc epsilon RII molecules were prepared having the mouse Fc epsilon RII lectin homology (carboxyl-terminal) region and the stalk region of either the related human Fc epsilon RII or the corresponding domain of Ly-49. Chimeric molecules using the former (alpha-helical coiled-coil) stalk supported normal binding of IgE although the Ly-49/Fc epsilon RII chimera failed to bind IgE. Taken together, the results indicate that high (approximately 10(8) M-1) affinity IgE binding results from interaction of multiple lectin domains with (presumably) symmetrical sites on the IgE molecule. Specificity for IgE is determined by the lectin domain although the binding avidity is determined by oligomerization through the coiled coil stalk.

Fc epsilon receptors.

Advances in our understanding of the molecular structure of Fc receptors have been made at a rapid pace. Details of how Fc receptors are involved in cell triggering, e.g. allergic mediator release from mast cells, and IgE synthesis are also continuing to be elucidated, although much work is still required. Recent highlights of investigations of mast-cell and lymphocyte IgE Fc receptors will be outlined.

Cognate interactions between helper T cells and B cells. VI. TGF-beta inhibits B cell activation and antigen-specific, physical interactions between Th and B cells.

TGF-beta is a 25-kDa homodimeric protein that has been shown to have multiple roles in the regulation of lymphocyte activation. Previous studies have shown that TGF-beta is an inhibitor of numerous T and B lymphocyte activities. This study shows that TGF-beta is able to inhibit Th- and mitogen-induced murine B cell proliferation, as well as mitogen-induced B cell cycle entry and immunoglobulin secretion. Acridine orange analysis established that TGF-beta inhibits the LPS-induced B cell transition from G0 to G1A phase of the cell cycle. Evaluation of the effects of TGF-beta on the interactions between Th and B cells showed that TGF-beta inhibited antigen-specific Th-B cell physical interactions. Flow cytometric data showed that the ability of TGF-beta to interfere with the formation of Th-B cell conjugates was not due to decreased expression of IgD, IgM, class I, class II, LFA-1, or ICAM1 by the B cell. Taken together, these data establish that TGF-beta is able to act at multiple sites within the immune system.

Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines.

Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN-gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL-5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells.

Cognate interactions between helper T cells and B cells. IV. Requirements for the expression of effector phase activity by helper T cells.

After activation with anti-CD3, activated Th (THCD3), but not resting Th, fixed with paraformaldehyde induce B cell RNA synthesis when co-cultured with resting B cells. This activity is expressed by Th of both Th1 and Th2 subtypes, as well as a third Th clone that is not classified into either subtype. It is proposed that anti-CD3 activation of Th results in the expression of Th membrane proteins that trigger B cell cycle entry. Kinetic studies reveal that 4 to 8 h of activation with anti-CD3 is sufficient for ThCD3 to express B cell-activating function. However, activation of Th with anti-CD3 for extended periods of time results in reduced Th effector activity. Inhibition of Th RNA synthesis during the anti-CD3 activation period ablates the ability of ThCD3 to induce B cell cycle entry. This indicates that de novo synthesis of proteins is required for ThCD3 to express effector function. The ability of fixed ThCD3 to induce entry of B cell into cycle is not due to an increase in expression of CD3, CD4, LFA-1, ICAM-1, class I MHC or Thy-1. Other forms of Th activation (PMA and A23187, Con A) also induced Th effector function. Furthermore, purified plasma membranes from anti-CD3 activated, but not resting Th, induced resting B cells to enter cycle. The addition of IL-4, but not IL-2, IL-5, or IFN-gamma amplified the DNA synthetic response of B cells stimulated with PM from activated Th. Taken together these data indicate that de novo expression of Th surface proteins on activated Th is required for Th to induce B cell cycle entry into G1 and the addition of IL-4 is required for the heightened progression into S phase.

Cognate interactions between helper T cells and B cells. II. Dissection of cognate help by using a class II-restricted, antigen-specific, IL-2-dependent helper T cell clone.

In order to determine the involvement of T-B cell contact vs lymphokine production in mediating B cell cycle entry and progression, Th cell clones "defective" in lymphokine production were cloned. Th-3.1 is one such clone that required IL-2 to produce significant levels of IL-4 and IFN-gamma. Unlike conventional Th clones, Th-3.1 induced B cell proliferation only in the presence of Ag and IL-2. In contrast to the absolute requirement of IL-2 for Th-3.1-induced B cell proliferation, IL-2 was not required for the formation of stable Th-3.1-B cell conjugates or Th-3.1-induced B cell entry into the G1 phase of the cell cycle. In the absence of IL-2 and under conditions that promoted Th-B cell interactions, Th-3.1 induced 10 to 20% of resting B cells to enter G1. B cell entry into the cell cycle was not inhibited by anti-lymphokine mAb or promoted by exogenous lymphokines, suggesting that endogenous lymphokine activity was not required for Th-3.1-induced G0 to G1 transition. The data suggested that the IL-2-independent induction of B cells into G1 by Th-3.1 was a cell contact-dependent event. Direct proof that Th-3.1-B cell contact was necessary for B cell cycle entry was provided by comparative in situ analysis of the RNA synthetic activity and the RNA content of B cells that were in physical contact with Th-3.1 or not in contact with Th-3.1. In situ autoradiography of RNA synthesis illustrated that a high frequency of B cells in contact with Th-3.1 expressed heightened RNA synthetic activity, whereas "bystander" B cells were less frequently induced into cycle. In situ laser cytometry of B cell size and total RNA content showed that B cells in physical contact with Th-3.1 had a higher RNA content and were larger than "bystander" B cells present in the same microcultures. This model system has allowed the dissection of T cell help into IL-2-dependent and IL-2-independent phases. Early cell contact-dependent events and B cell cycle progression into G1 were IL-2 independent, whereas the production of lymphokines (IL-4, IFN-gamma) by Th-3.1 and Th-3.1-induced B cell proliferation was IL-2 dependent.

Cognate interactions between helper T cells and B cells. III. Contact-dependent, lymphokine-independent induction of B cell cycle entry by activated helper T cells.

An Ag-specific, IL-2-dependent Th clone induced the growth of B cells in a class II-restricted, Ag-specific, IL-2-dependent manner. The formation of stable Th-3.1-B cell conjugates was restricted by Ag and class II MHC. After activation of Th-3.1 by insolubilized anti-T3 (Th-3.1T3), Th-3.1T3 induced the growth of B cells in a class II unrestricted, Ag nonspecific manner. The formation of stable conjugates between Th-3.1T3 and B cells was also class II unrestricted and Ag nonspecific. Although the interaction of Th-3.1T3 and B cells was class II unrestricted, the interaction was inhibited by the combination of anti-IA and anti-IE mAb. This suggested that monomorphic domains of class II MHC molecules were involved in Th-3.1T3-B cell interaction. Fixed Th-3.1T3 but not fixed resting Th-3.1 induced B cell cycle entry, as measured by an increase in B cell RNA synthesis. Trypsin-treatment of Th-3.1T3 before fixation reduced their ability to activate B cells, indicating that cell surface proteins on Th-3.1T3 were required for enhanced B cell RNA synthesis. Anti-IL-4, anti-IL-2R, or anti-IFN-gamma did not affect the ability of Th-3.1T3 to induce heightened B cell RNA synthesis. Progression into S phase by B cells activated with fixed Th-3.1T3 was supported by the addition of soluble factors. When stimulated with fixed Th-3.1T3, EL4 supernatant (SN) enhanced B cell DNA synthesis. Depletion of IL-4, but not IL-2, from EL4 SN ablated its supportive capabilities. IL-4 alone was completely ineffective in supporting entry into S phase. Therefore, IL-4 and another activity(ies) in EL4 SN were necessary for B cell cycle progression into S phase. Taken together, these data suggest that after Th activation, Th cell surface proteins are expressed that mediate the binding of Th to B cells via recognition of nonpolymorphic domains of class II MHC molecules. Contact of Th-3.1T3 with B cells, not lymphokines, results in the entry of B cells into the cell cycle and heightened B cell lymphokine responsiveness. The addition of exogenous lymphokines supports the progression of Th-3.1T3-activated B cells into S phase.

A cell surface ELISA to detect interleukin-4-induced class II MHC expression on murine B cells.

A sensitive cell surface ELISA is described to detect interleukin-4 (IL-4)-induced major histocompatibility complex class II expression on murine B cells. The ELISA is performed on B cells cultured for 16 h in the presence of purified IL-4 or T cell-derived supernatants. A sandwich technique employing a biotin-conjugated monoclonal anti-I-E antibody followed by avidin-conjugated alkaline phosphatase is used to detect enhanced I-E MHC antigen expression on the B cells. Detection of I-E induced by both purified IL-4 and a T cell-derived supernatant was highly reproducible and sensitive using this method. Other lymphokines were found to be negative for the induction of I-E. The assay is easily performed and many sample supernatants can be rapidly screened for IL-4 activity. In addition, a general method for the removal of endogenous inhibitors of lymphokines from complex supernatants is described.