Arieh Licht

Selective inhibition of the FcεRI‐induced de novo synthesis of mediators by an inhibitory receptor

Aggregation of the type 1 Fcε receptors (FcεRI) on mast cells initiates a network of biochemical processes culminating in secretion of both granule‐stored and de novo‐synthesized inflammatory mediators. A strict control of this response is obviously a necessity; nevertheless, this regulation is hardly characterized. Here we report that a prototype inhibitory receptor, the mast cell function‐associated antigen (MAFA), selectively regulates the FcεRI stimulus–response coupling network and the subsequent de novo production and secretion of inflammatory mediators. Specifically, MAFA suppresses the PLC‐γ2–[Ca2+]i, Raf‐1–Erk1/2, and PKC–p38 coupling pathways, while the Fyn–Gab2‐mediated activation of PKB and Jnk is essentially unaffected. Hence, the activities of several transcription/nuclear factors for inflammatory mediators (NF‐κB, NFAT) are markedly reduced, while those of others (Jun, Fos, Fra, p90rsk) are unaltered. This results in a selective inhibition of gene transcription of cytokines including IL‐1β, IL‐4, IL‐8, and IL‐10, while that of TNF‐α, MCP‐1, IL‐3, IL‐5, or IL‐13 remains unaffected. Taken together, these results illustrate the capacity of an immunoreceptor tyrosine‐based inhibitory motif‐containing receptor to cause tight and specific control of the production and secretion of inflammatory mediators by mast cells.

Regulation of mast cells’ secretory response by co-clustering the Type 1 Fcϵ receptor with the mast cell function-associated antigen

The mast cell function‐associated antigen (MAFA) is a type II membrane glycoprotein first identified on rat mast cells and basophils. Clustering MAFA inhibits these cells’ secretory response to the type 1 Fcϵ receptor (FcϵRI) stimulus. To quantitatively characterize this inhibition and its dependence on MAFA‐FcϵRI co‐clustering, we investigated the secretory response of rat mucosal‐type mast cells of the RBL 2H3 line carrying an IgE class, 2,4 dinitrophenyl (DNP) specific monoclonal antibody to DNP‐conjugated Fab and F(ab′)2 fragments of (1) mouse IgG, and (2) of the MAFA‐specific, monoclonal antibody G63. The first reagent clusters FcϵRI‐IgE complexes into oligomers by reacting with the DNP residues. The DNP conjugated G63 Fab and F(ab′)2 fragments, additionally aggregate MAFA and form FcϵRI‐IgE‐MAFA co‐clusters. All experiments using these ligands were performed in the absence or presence of an excess of intact mAb G63, which clusters MAFA molecules. Empirical Hill functions were used to relate the secretory response of mast cells to the equilibrium concentrations of FcϵRI‐IgE or MAFA clusters and co‐clusters calculated as function of the employed ligands concentrations. This analysis of the experimental results indicates that co‐clustered MAFA molecules have a markedly higher inhibitory capacity than MAFA‐clusters alone. The molecular basis of the enhanced inhibition observed upon co‐clustering MAFA with the FcϵRI is most probably the increased concentration of the inhibitory cell components in the immediate proximity of the activation coupling elements.

A glycolipid-specific monoclonal antibody modulates Fcϵ receptor stimulation of mast cells

Abstract In an effort to identify membrane components participating in coupling stimulus to secretion in mast cells, monoclonal antibodies were produced from spleen cells of mice immunized with plasma membranes isolated from rat mast cells of the RBL-2H3 line. The resultant mAbs were screened by their capacity to modulate the secretory response of these cells to crosslinking of their type 1 Fcϵ receptor (Fc ϵ RI). Following this scheme, we obtained a hybridoma designated B17, which secretes an IgM-class mAb (B17) that binds to and modulates secretion from RBL-2H3 cells. By immunoblotting, B17 was shown to bind to a membrane component of low molecular weight, later identified as a glycolipid. While B17 partially inhibits IgE binding to RBL-2H3 cells, no noticeable inhibition of B17 binding by IgE was observed. mAb B17 does not cause any secretory response on its own, and its modulatory effect on Fc ϵ RI-mediated secretion is bimodal: it either enhances or inhibits secretion, depending on the B17 dose and also on the nature and dose of the agent used for crosslinking the Fc ϵ RI. When secretion was induced by IgE and suboptimal or optimal doses of multivalent antigen, B17 (2–80 nM) caused an increase in secretion. However, higher doses of B17 (>150nM) inhibited secretion. Secretion induced by supraoptimal doses of antigen, or by the Fc ϵ RI-specific mAb F4 was inhibited by B17 at all the dose range tested (2–200 nM). In contrast, B17 had no effect on secretion induced by Ca 2+ ionophores. These results demonstrate that Fc ϵ RI function is modulated by a mAb binding to a membrane glycolipid.

An IgE‐dependent secretory response of mast cells can be induced by a glycosphingolipid‐specific monoclonal antibody

The signal transduction pathway of the type 1 Fcϵ receptor (FcϵRI) has been proposed to be spatially constrained to plasma membrane microdomains enriched in glycosphingolipids and cholesterol. These domains are proposed to serve as platforms that enhance the efficiency of the antigen‐receptor stimulus‐response coupling process. Here we describe a monoclonal antibody (mAb) designated 2B5, raised by immunizing mice with rat mucosal‐type mast cell (line RBL‐2H3) membranes, which binds to glycosphingolipids and causes a dose‐dependent secretory response of these cells. This secretory response to mAb 2B5 requires binding of IgE to the FcϵRI on these cells, although direct interactions between IgE and mAb 2B5 are excluded. The bound IgE‐ or FcϵRI‐specific mAb did not affect binding of mAb 2B5 or its Fab fragments to the RBL‐2H3 cells and only a limited interference with the binding of IgE to the FcϵRI by mAb 2B5 was observed. Binding of mAb 2B5 to the RBL‐2H3 cells induced a distribution of fluorescently labeled IgE similar to that produced by antigen‐induced aggregation of the IgE‐FcϵRI. Thus we suggest that mAb 2B5 binds to cell surface glycosphingolipids that are probably associated with the FcϵRI‐IgE complexes and causes their aggregation, thereby initiating the cascade leading to the cells secretory response.

Mast cell stimulation by co-clustering the type I Fcε-receptors with mast cell function-associated antigens

Abstract The secretory response of rat mucosal-type mast cells (line RBL 2H3) to stimuli produced by clustering or co-clustering two of its membranal components; the type I Fce receptor and the mast cell function associated antigen (MAFA) was investigated. The primary reagents employed for this purpose were Fab fragments of the monoclonal antibodies J17 and G63 specific to the above respective proteins. The Fabs were then aggregated by F(ab′)2 fragments of mouse IgG specific goat antibodies. This reaction was assumed to yield predominantly three different bivalent clustering reagents. Namely, dimers of the FceRI specific (J17-Fab)2; dimers of the MAFA specific, (G63-Fab)2 and bispecific (J17-Fab-G63-Fab) dimers. The observed cellular secretory response was analyzed by employing a model which accounts for the clustering and co-clustering of FceRIs and MAFAs by the above protocols. Results of this analysis provided evidence that at least some of the MAFA molecules are physically associated with the FceRI. As a consequence, clustering of MAFA and FceRI by bispecific J17-Fab-G63-Fab dimers induces secretion at comparatively low concentrations of these reagents, though with a significantly lower maximal response than that caused by the respective monospecific reagent (J17-Fab)2. This result most likely reflects the inhibitory capacity of MAFA-FceRI interaction.

Thermodynamic and spectroscopic comparison of the binding sites of the mouse myeloma protein 315 and of its light chain dimer

The homogeneous IgA secreted by MOPC 315 tumor was shown to bind specifically some nitroaromatic derivatives and other electron acceptor aromatic compounds [I]. A detailed investigation of the combining site of the intact molecule (I-IL) by kinetic mapping resolved several subsites of binding, each contributing elementary interactions with defined portions of the hapten [2,3]. These subsites are: the nitroaromatic binding subsite (Sr), the first and second hydrophobic subsites (Sa and Ss), and electrostatic positively charged subsite (S4). Model building of the variable region of this protein, enabled a three dimensional analysis of the binding site which agreed with the available thermodynamic and kinetic data [4]. Recently it was found that the light chain of protein 3 15 also binds e-N(2,4dinitrophenyl) lysine (DNPL) specifically, though with significantly lower affinity [5]. The light chain was shown to exist in neutral aqueous solutions as a dimer &) which binds homogeneously two moles of DNPL (K = 6.3 X lo3 M-r at 4’C). This is the first case where the light chain of a homogeneous immunoglobulin is shown to have a significant and specific binding capacity for the same ligand as its parent molecule, providing an experimental approach to examine the role of the light chain in the architecture and specificity of the antibody

Positive cooperativity in the hapten binding by the VL dimer of protein 315.

Abstract The hapten binding behavior of the dimer of the variable domains, (V l )2, derived from the light chains of protein 315 was investigated by difference absorption spectrophotometric titrations. The stoichiometry of binding of e-N-(2,4- dinitrophenyl)- l -lysine (DNPL) and 4-(α-N-alanine)-7-nitrobenz-2-oxa-1,3-diazole is two haptens per (V l )2. Positive cooperativity was observed in this binding. Such positive cooperativity has been reported previously for the light chain dimers of protein 315 with their native interchain disulfide bridge preserved or cleaved by reduction and alkylation. The hapten binding was analysed as in the previous cases according to the allosteric model of Monod et al. (1965). This model assumes that the protein exists in two conformations, and the equilibrium between them shifts upon hapten binding. The parameters of the hapten binding and of the allosteric transition of (V l )2 are similar to those of L2 ncov showing that the C l domains play a rather limited role in the behavior of the latter protein. In contrast, these parameters are drastically different from those reported for the L2 cov. This constitutes a further illustration of the importance of the disulfide bond in affecting the proteins conformation and hapten binding. The detailed analysis of the titrations of (V l )2 and of L2nocov with DNPL led to the calculation of the difference absorption spectra between the DNPL complexes formed by each of the two conformers of either protein and the free hapten. The distinct dissimilarity of these two difference spectra illustrates the different environments of the dinitrophenyl ring in the binding sites of each conformer.

Pulse radiolysis kinetics of the reaction of the hydrated electron and the carboxyl anion radical with Pseudomonas aeruginosa cytochrome c-551

The kinetics of the reaction of hydrated electron (e−aq) and carboxyl anion radical (CO⨪2) with Pseudomonas aeruginosa ferricytochrome c-551 were studied by pulse radiolysis. The rate of reaction of e−aq with the negatively charged ferricytochrome c-551 (17 nM−1 · s−1) is significantly slower than the larger positively charged horse heart ferricytochrome c (70 nM− · s−). This difference cannot be explained solely by electrostatic effects on the diffusion-controlled reactions. After the initial encounter of e−aq with the protein, ferricytochrome c-551 is less effective in transferring an electron to the heme which may be due to the negative charge on the protein. The charge on ferricytochrome c-551 is estimated to be −5 at pH 7 from the effect of ionic strength on the reaction rate. A slower relaxation (2 · 104 s−1) observed after fast e−aq reduction is attributed to a small conformational change. The rate of reaction of CO⨪2 with ferricytochrome c-551 (0.7 nM−1 · s−) is, after electrostatic correction, the same as ferricytochrome c, indicating that the steric requirements for reaction are similar. This reaction probably takes place through the exposed heme edge.

On the conformation of antibodies in the presence and absence of antigen (small-angle X-ray studies)

The conformations of different IgG antibodies were studied before and after interaction with antigen (hapten). In every case a strong change of the conformation was observed. Binding of hapten caused a decrease of the radius of gyration by 2 to 8% and a decrease of the volume by 3 to 10%, depending on the degree of saturation with hapten. Two IgG antibodies (anti-poly-d-alanyl) were split by enzymes into fragments which contain one binding site (Fab′) and two binding sites (Fab′)2, respectively, for hapten. No changes of conformation were observed with these fragments upon the interaction with hapten. These findings lead to the conclusion that the conformation change does not take place within the area of the combining site but relatively far away, at the area of the hinge region and/or the Fc-fragment.