Increasing the serum persistence of an IgG fragment by random mutagenesis.

The major histocompatibility complex (MHC) class I-related receptor FcRn is involved in regulating serum gammaglobulin (IgG) levels in mice. With the aim of increasing the serum half-life of a recombinant murine Fc gamma 1 fragment, the affinity for binding to FcRn at pH 6.0 has been increased by random mutagenesis of Thr252, Thr254, and Thr256 followed by selection using bacteriophage display. These residues were chosen as they are in proximity to the FcRn-IgG (Fc) interaction site. Two mutants with higher affinity (due to lower off-rates) than the wild-type Fc have been isolated and analyzed in pharmacokinetic studies in mice. The mutant with the highest affinity has a significantly longer serum half-life than the wild type fragment, despite its lower off-rate from FcRn at pH 7.4. The results provide support for the involvement of FcRn in the homeostasis of serum IgGs in mice. The indications that a homologous FcRn regulates IgG levels in humans suggest that this approach has implications for increasing the serum persistence of therapeutic antibodies.

Antibodies against glutaraldehyde-modified albumin in normal mouse sera.

An antigen-coated plate radioimmunoassay was used to detect antibodies against homologous glutaraldehyde-modified albumin (in monomeric or polymeric form) in normal mouse sera. Mouse antibodies reacted also with heterologous glutaraldehyde-treated albumin; for instance human albumin. Immunoelectrophoresis of purified anti-albumin antibodies and adsorption experiments on protein A-Sepharose 4B gel indicated that mouse antibodies belong mainly to the IgG class. The ability of mouse anti-albumin antibodies to react also with pyridinium compounds suggested that such structures are probably part of the new antigenic determinants induced in mouse albumin by glutaraldehyde treatment. It was assumed that anti-albumin antibodies have a physiologic role in the recognition and removal from the circulation of in vivo 'aged' mouse albumin.

Anti-albumin antibodies in normal rabbit sera.

Antibodies reacting with homologous glutaraldehyde (GA)-modified albumin were demonstrated in normal rabbit sera (NRS) by passive hemagglutination and direct binding assay. The ability of rabbit anti-albumin antibodies (AAA) to react with homologous GA-modified albumin was found to increase with the degree of albumin polymerization. AAA did not react with GA-treated monomeric albumin. It was proved that rabbit AAA are not species-specific since they react also with heterologous GA-polymerized albumin. The possible role of AAA in the catabolism of in vivo aged albumin molecules, antigenically similar with GA-induced albumin polymers, is discussed.

A method for the detection and quantitation of Fc receptor sites on cells.

A new rosette technique for identification of Fc-receptor-bearing cells is based on the ability of sheep erythrocytes coated with protein A of Staphylococcus aureus (ES) to form rosettes with cells treated with monomeric IgG or aggregated IgG. The IgG is attached to lymphocytes through its Fc region and binds to a trypsin-resistant but pronase-sensitive receptor (considered an Fc receptor). The ES rosette technique facilitates studies of the interaction of IgG with Fc receptor sites; the binding of any IgG preparation reacting with protein A of Staphylococcus aureus (SpA) can be studied by the technique. Inhibition of rosette formation by SpA was used for quantitation of IgG fixed to the cell surface (that is, the number of Fc receptors/cell). The sensitivity of the method permits quantitation of less than 10(5) IgG molecules bound to the Fc receptors. The relative affinity constant between Fc receptors and IgG ligands was estimated by plotting the percentage of ES rosettes as a function of IgG concentration and calculating the reciprocal of the IgG concentration giving half the maximal number of ES rosettes.

Immunohistochemistry of thyroid hormones as a functional parameter in thyroid pathology.

The authors study by means of immunoperoxidase method the pattern of thyroglobulin, triiodothyronine and thyroxine distribution in 58 cases of thyroid disorders: 15 euthyroid goiters, 10 Graves' disease, 7 Hashimoto's thyroiditis, 11 folliculo-papillary carcinomas (6 primary tumors and 5 lymph node metastases), 8 follicular carcinomas, 4 anaplastic carcinomas and 3 medullary carcinomas. Thyroglobulin, triiodothyronine and thyroxine were present in most of the thyroid disorders, excepting anaplastic and medullary carcinomas. Thyroglobulin and thyroxine were localized both in the follicular epithelium and in the colloid, whereas triiodothyronine was present especially in the follicular cells. The thyroid hormones distribution in benign lesions is rather similar. In carcinomas, the pattern of thyroglobulin, triiodothyronine and thyroxine is more heterogeneous, but generally the triiodothyronine distribution is similar to that of thyroglobulin. In some carcinomas, triiodothyronine and thyroxine showed a weak or negative immunostaining. The immunoperoxidase method is a valuable tool in the study of functional disturbances in the thyroid pathology and in the diagnosis of thyroid carcinoma metastases as well. Positive thyroid hormones staining clearly indicates the thyroid origin of metastases.

Delineation of the amino acid residues involved in transcytosis and catabolism of mouse IgG1.

The MHC class I-related receptor, FcRn, is involved in both the transcytosis of serum gamma-globulins (IgGs) and in regulating their serum persistence. The interaction site of FcRn on the Fc region of rodent IgG has been mapped to residues at the CH2-CH3 domain interface using site-directed mutagenesis and x-ray crystallographic analyses. In the current study, the role of individual residues (H310, H433, and N434) at this interface in mediating the Fc-FcRn interaction has been investigated using recombinant, mutated Fc hinge fragments derived from mouse IgG1. In addition, two highly conserved Fc histidines (H435 and H436) have been mutated to alanine, and the resulting mutated Fc hinge fragments were analyzed in both transcytosis and pharmacokinetic studies in mice and in competition binding assays using recombinant, soluble FcRn. The analyses indicate that mutation of H310, H435, and, to a lesser extent, H436 to alanine results in reduced activity of the Fc hinge fragments in both in vivo and in vitro assays. Thus, in addition to the previously defined role of 1253 in the FcRn-IgG interaction, these histidines play a key role in mediating the functions conducted by this Fc receptor. The effects of these mutations on binding of Fc hinge fragments to staphylococcal protein A have also been analyzed and demonstrate a partial, but not complete, overlap of the FcRn and staphylococcal protein A interaction sites on mouse IgG1.

Localization of the site of the IgG molecule that regulates maternofetal transmission in mice.

Site-directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the mouse IgG1 (mIgG1) molecule that is involved in the intestinal transfer of recombinant Fc hinge fragments in neonatal mice. This site encompasses Ile-253, His-310, Gln-311, His-433 and Asn-434, localized at the CH2-CH3 domain interface and overlapping with the staphylococcal protein A-binding and catabolic sites. In the present study, the effect of these mutations on the maternofetal transfer of Fc hinge fragments has been studied. Experiments to analyze transfer of radiolabeled Fc hinge fragments from the circulation of 15-18 day pregnant mice to fetuses in utero demonstrate that the mutations affect the maternofetal transmission in a way that correlates closely with the effects of the mutations on intestinal transfer and catabolism. The studies indicate that the neonatal Fc receptor, FcRn, is involved in transcytosis across both yolk sac and neonatal intestine in addition to the regulation of IgG catabolism.

Effect of protein A and its fragment B on the catabolic and Fc receptor sites of IgG.

Radiolabeled protein A from Staphylococcus aureus (SpA) injected i.v. into mice and rabbits forms a soluble [(IgG)2-(SpA)1]2 complex (Mr = 684 000) which is identical in composition to that formed by SpA in vitro with an equivalent amount or an excess of IgG. A soluble rabbit IgG-SpA complex injected into a mice or rabbits dissociates completely in vivo and a new complex is formed with the IgG of the recipient animal. The half-life of SpA administered to a mouse or a rabbit is therefore the half-life of the IgG-SpA complex formed in vivo. In mice and rabbits the half-life of the complexes formed is 9 and 30 h, respectively, whereas the half-life of rabbit IgG in these animals is 106 and 153 h, respectively. Fragment B of SpA (fSpA) reacts with IgG of mouse and rabbit and forms an (IgG)1-(fSpA)1 complex. Complexes of identical composition are formed if fSpA is injected i.v. into mice and rabbits. The half-life of the complexes in mice and rabbits are much shorter than those of the corresponding free IgG in these animals (up to 15 times). This result suggests that the binding of fSpA to the CH2 and the CH3 domains of IgG alters the function of the site, which controls the catabolism of IgG and is located in the CH2 domain. By contrast, fSpA does not change the Fc receptor-binding site of IgG, indicating that the Fc receptor site and the catabolic site are unrelated to each other.

Identification and function of neonatal Fc receptor in mammary gland of lactating mice.

In addition to its proposed function in regulating serum IgG levels, the MHC class I-related neonatal Fc receptor (FcRn) is known to play a role in IgG transfer across rodent yolk sac and neonatal intestine. In contrast to humans, for which transplacental transfer of IgG appears to be the only mechanism of maternal IgG delivery, the transmission of IgG in mice occurs both antenatally (yolk sac) and neonatally (transport from mother's milk across intestinal epithelial cells). In the current study, a possible role for FcRn in regulating IgG transfer into milk has been investigated. FcRn has been shown to be present in functional form in the mammary gland of lactating mice, and is localized to the epithelial cells of the acini. Analysis of the transfer of Fc fragments and IgG which have different affinities for FcRn indicate that, unexpectedly, these proteins are transferred in inverse correlation with their binding affinity for FcRn. Thus, in the lactating mammary gland FcRn appears to play a role in recycling IgG in a mode that may have relevance to FcRn trafficking during the maintenance of constant serum IgG levels.

IgG-binding sites on macrophage cell membrane. II. Mobility of Fc receptors induced by the interaction with their corresponding IgG ligands.

Mouse peritoneal macrophages were charged with IgG molecules in monomeric (mIgG), heat-aggregated (agIgG) or antigen-complexed (acIgG) form. Upon exposure to 37 degrees C, all bound IgG ligand types are redistributed on the cell surface due to the mobilization of their corresponding Fc receptor (FcR). The major findings regarding the fate of FcR on macrophages bearing IgG ligands are as follows: (a) the FcR involved in the binding of cytophilic molecules has a slow movement on the cell membrane and forms patches but never caps, while the opsonic type of FcR is rapidly capped; (b) the mobility of IgG-binding sites was temperature-dependent and was affected differently by sodium azide; this metabolic inhibitor enhances the disappearance of mIgG from the cell surface but decreases the capping and the disappearance of polymeric ligands; (c) both FcR types are probably ingested when complexed with specific ligand, and consequently, the rebinding of homologous IgG molecules is reduced, the clearing induced by agIgG or acIgG binding being much more extensive; and (d) cells cleared of their opsonic types of FcR are able to regenerate the receptor molecules with 8 h of incubation at 37 degrees C.

IgG-binding sites on macrophage cell membrane. I. Identification of two distinct Fc receptors on mouse peritoneal macrophages.

Evidence is presented concerning the existence on mouse peritoneal macrophages of two separate and distinct Fc receptors, one for cytophilic monomeric IgG (mIgG) and the other for polymeric IgG. The latter Fc receptor recognizes both heat-aggregated IgG and antigen-complexed IgG. The major findings of our studies are: (a) the different susceptibility of the two Fc receptor types by pronase, trypsin or phospholipase C; (b) the independent modulation of these two binding sites on the cell membrane; (c) the inability of mIgG to inhibit the binding of particulate antigen-complexed IgG ligand; (d) the ability of mIgG molecules which are devoid of the cytophilic property to attach to the macrophage surface upon their polymerization induced by heating or antigen. The results are discussed in terms of "cytophilic" and "opsonic" Fc receptor types which may provide different functional abilities for normal macrophages.

Effect of protein A of Staphylococcus aureus on the binding of monomeric and polymeric IgG to Fc receptor-bearing cells.

The effect of protein A of Staphylococcus aureus (SpA) on the binding of rabbit IgG to the Fc receptors of mouse lymphocytes and macrophages was found to correlate with the aggregation of the IgG ligands. After binding anti-erythrocyte IgG complexed with SpA, the cells were able to attach to and kill erythrocyte indicator cells with a higher efficiency than lymphoid cells treated with anti-erythrocyte IgG alone. The amount of anti-peroxidase IgG which can be bound to effector cells was not changed by reaction with SpA. In contrast, the binding to cells of IgG-coated erythrocytes and of anti-peroxidase IgG complexed with peroxidase was substantially reduced by reaction with SpA. The results are compatible with the presence of two distinct Fc receptors, one for cytophilic monomeric IgG and another for polymeric (antigen-complexed) IgG.

Comparative studies of rat IgG to further delineate the Fc:FcRn interaction site.

Recent data have indicated that the MHC class I-related receptor, FcRn, regulates the half-lives of serum IgG in addition to its known role in transferring IgG from mother to young. In the current study, the activity of rat IgG (rIgG) isotypes in FcRn-mediated functions has been analyzed. The serum half-life and maternofetal transfer in mice decreased in the order rIgG2a > rIgG1 > rIgG2c > rIgG2b. This decrease in activity correlates well with reduced binding affinity for soluble mouse FcRn, and site-directed mutagenesis of a recombinant Fc-hinge fragment has been used to investigate the molecular basis for the differences in activities of the rIgG. Analysis of the serum half-lives of the mutated Fc-hinge fragments demonstrated that, in addition to Ile253, His310, His435 and His436 that were identified in earlier studies, amino acids at positions 257, 307 and 309 play a role in building the FcRn interaction site of IgG. The study also excludes the involvement of amino acids in a fourth loop located at the CH2-CH3 domain interface that encompasses residues 386-387 in FcRn binding. Sequence differences at positions 257, 307 and 309 between rIgG most likely account for the reduced affinity of rIgG2b and IgG2c relative to rIgG1 and rIgG2a for binding to FcRn.

Effects of B4 Coxsackievirus infection on cell-mediated immunity, natural cytotoxicity and interleukin-1 production by spleen cells, in mice.

By using CBA inbred mice as responders, the influence of in vivo B4 Coxsackievirus infection (in two separate experimental procedures) on anti-virus cell-mediated immunity (CMI), natural killer (NK) cell activity and lipopolysaccharide (LPS) induced interleukin-1 (IL-1) production in spleen cells were studied. Both experimental infection schedules were able to induce a "positive" CMI response. NK cell activity was also significantly stimulated, especially by the administration of high virus amounts/inoculum, with frequent injections. On the contrary, LPS-dependent IL-1 synthesis increased particularly following application of the other experimental schedule (low virus amounts, few injections). Some aspects concerning CMI, NK and IL-1 relationships, during several enteroviral diseases, are discussed.

Functional expression of the MHC class I-related receptor, FcRn, in endothelial cells of mice.

Our recent data indicate that the MHC class I-related receptor, FcRn, plays a role in regulating serum IgG levels, in addition to its known role in transferring IgG from mother to young. In the current study, the distribution of FcRn in adult mice has been investigated using several approaches. First, tissue distribution of anti-FcRn F(ab')2, murine IgG1 and recombinant, IgG1-derived Fc-hinge fragments has been analyzed, and these FcRn binding proteins localize predominantly in skin and muscle with lesser amounts in liver and adipose tissue. Second, histochemical analyses of muscle and liver with anti-FcRn F(ab')2 indicate that FcRn is expressed in the endothelium of small arterioles and capillaries, but not in larger vessels such as the central vein and portal vasculature. Third, immunoprecipitation and immunofluorescence studies of cultured murine endothelial cells show that functional FcRn is expressed in these cells, and is located within vesicular structures in the cytosol and not on the membrane. Taken together the data demonstrate that FcRn is expressed in functionally active form in endothelial cells, indicating that these cells are a possible site at which serum IgG homeostasis is maintained.