The interaction of IgG subclasses with solubilized Fc receptors of rat basophilic leukemia cells

Rat basophilic leukemia (RBL) cells carry two surface glycoprotein molecules named R (or alpha) and H which, when detergent solubilized, bind to rat IgE-Sepharose. The same two molecules also bind to rat IgG-Sepharose but with a lower affinity. R is a component of the high affinity Fc receptor for IgE. In the present study the inhibition of the binding of R and H to rat IgG-Sepharose by various homologous and heterologous immunoglobulins was used to assess their relative affinities for the two receptor molecules. Ranking the rat immunoglobulins in order of their affinities for the R receptor yielded: IgE much greater than IgG2a greater than IgG1 greater than IgG2b; and for H: IgE greater than IgG2b greater than IgG1 greater than IgG2a. Rat IgG2c inhibited the binding of both R and H but a precise ranking could not be assigned. Conclusive evidence has been obtained for the Fc specificity of these interactions. The affinities of the mouse IgG subclass/R interactions can be ranked: IgG1 greater than IgG2a greater than IgG2b; and for the H receptor: IgG1 greater than IgG2b greater than IgG2a. All of the mouse proteins and other heterologous IgGs, such as those of sheep, goat, equine and rabbit origin, interacted considerably more strongly with H than with R. No interaction with mouse IgG3 could be detected under the conditions tested.     

Over-expression of the bovine FcRn in the mammary gland results in increased IgG levels in both milk and serum of transgenic mice

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from catabolism and is also responsible for IgG absorption in the neonatal small intestine. However, whether it mediates the transfer of IgG from plasma to milk still remains speculative. In the present study, we have generated transgenic mice that over-express the bovine FcRn (bFcRn) in their lactating mammary glands. Significantly increased IgG levels were observed in the sera and milk from transgenic animals, suggesting that the over-expressed bFcRn could bind and protect endogenous mouse IgG and thus extend its lifespan. We also found that injected human IgG showed a significantly longer half-life (7-8 days) in the transgenic mice than in controls (2.9 days). Altogether, the data suggested that bFcRn could bind both mouse and human IgG, showing a cross-species FcRn-IgG binding activity. However, we found no selective accumulation of endogenous mouse IgG or injected bovine IgG in the milk of the transgenic females, supporting a previous hypothesis that IgG was transported from serum to milk in an inverse correlation to its binding affinity to FcRn.     

Studies on the Interaction of Human Immunoglobulin E with Rat Mast Cells

The present study was undertaken to determine if human IgE immunoglobulin could be shown to bind to rat peritoneal mast cells and to establish whether binding is a prerequisite for the cellular alterations taking place during the rat mast cell test. It was found that sensitization of rat mast cells occurs very rapidly in the presence of human IgE immunoglobulin. Thus, incubation of the IgE immunoglobulin at 37°C for 3 min with rat mast cell suspension and washing the suspension once with medium 199 led to morphological changes in the mast cells on challenge with anti-human IgE immunoglobulin. However, attempts to correlate longer periods of incubation time with increased sensitization or show the extent of binding by multiple washings of the cell suspension were unsuccessful due to the lability of the rat mast cells. On the other hand, significant uptake of IgE immunoglobulin was observed by the mast cells after exposing the cell suspension to (a) radiolabelled ¹²⁵I-IgE immunoglobulin for 3 min at 37°C and washing the suspension and (b) IgE immunoglobulin for 3 min at 37°C, washing the cell suspension and challenging the cells with FITC-labelled anti-human IgE immunoglobulin. At 3 min exposure time, human IgE immunoglobulin was not bound by neutrophils or eosinophils nor was human IgG immunoglobulin bound by mast cells.     

The binding of proteins to isolated enterocytes from the small intestine of the neonatal rat.

IgG binds specifically to isolated jejunal enterocytes but not to ileal enterocytes; maximum binding occurred at pH 6. The ability of jejunal enterocytes to bind IgG was reduced to low levels at 20 days of age and was lost at 24 days. Human and rat IgG were bound specifically in similar amounts; human IgG displaced rat IgG with identical efficiency to rat IgG (ED50 = 50 nM). Much less bovine and sheep IgG were bound to enterocytes and the ED50s for these proteins were 150 nM and 2.5 microM, respectively. Rat IgG bound to jejunal enterocytes with high affinity (13.21 x 10(6)M-1) and to 4.83 x 10(6) sites per cell. Receptor protein was estimated to represent 0.18% of total cell protein. These observations are discussed in relation to the results of in vivo IgG transmission studies. It is estimated that the IgG transport mechanism, operating at maximum efficiency, requires that available IgG receptors would come into use once to twice per hour.     

Studies on the Interaction of Human Immunoglobulin E with Rat Mast Cells

The present study was undertaken to determine if human IgE immunoglobulin could be shown to bind to rat peritoneal mast cells and to establish whether binding is a prerequisite for the cellular alterations taking place during the rat mast cell test. It was found that sensitization of rat mast cells occurs very rapidly in the presence of human IgE immunoglobulin. Thus, incubation of the IgE immunoglobulin at 37°C for 3 min with rat mast cell suspension and washing the suspension once with medium 199 led to morphological changes in the mast cells on challenge with anti-human IgE immunoglobulin. However, attempts to correlate longer periods of incubation time with increased sensitization or show the extent of binding by multiple washings of the cell suspension were unsuccessful due to the lability of the rat mast cells. On the other hand, significant uptake of IgE immunoglobulin was observed by the mast cells after exposing the cell suspension to (a) radiolabelled ¹²⁵I-IgE immunoglobulin for 3 min at 37°C and washing the suspension and (b) IgE immunoglobulin for 3 min at 37°C, washing the cell suspension and challenging the cells with FITC-labelled anti-human IgE immunoglobulin. At 3 min exposure time, human IgE immunoglobulin was not bound by neutrophils or eosinophils nor was human IgG immunoglobulin bound by mast cells.     

Protection of mice against Mycoplasma pulmonis infection using purified mouse immunoglobulins: comparison between protective effect and biological properties of immunoglobulin classes.

Purified mouse immunoglobulins with specific activity against Mycoplasma pulmonis were examined for their ability to protect mice against intranasal (i.n.) infection with this organism. The survival of mycoplasmas in the respiratory tract of mice inoculated i.n. with M. pulmonis mixed with either IgG1, IgGa, IgG2b or IgA antibodies was less than that seen in control animals inoculated with mycoplasmas mixed with normal mouse serum. The ways in which the immunoglobulin fractions could have mediated resistance were examined in vitro. IgG1, IgG2a and IgG2b antibody fractions killed M. pulmonis in the presence of guinea-pig complement, and also promoted the phagocytosis of M. pulmonis by mouse lung macrophages. In contrast, IgA antibody did not appear to be either mycoplasmacidal or opsonic.     

Affinity of immunoglobulin for heterologous tissue mast cells. A study with the fluorescent antibody method

A double layer immunofluorescence method was used to explore the affinity of immunoglobulins and immunoglobulin fragments for the mast cells of the mouse tongue. Human IgG but not IgA or IgM showed such an affinity as revealed by fluorescence of globulin attached to the mast cell surface and to the mast cell granules. This affinity was found also in isolated H(γ) chains but not in the light chains of IgG. After papain digestion, the Fab and Fc fragments obtained showed no affinity for mouse tongue mast cells though the 5S fragment obtained after pepsin digestion retained activity. The human immunoglobulin sub-class IgG₂ lacks the ability to bind to mouse tongue mast cells. In guinea-pig serum, γ₁- but not γ₂-globulin showed an affinity for mouse tongue mast cells. It was suggested that a specific attachment site for mouse tongue mast cell surface receptors was present on the γ chain of human IgG and that this site was in a position susceptible to attck by papain hydrolysis.     

Isolation of a 30 kDa immunoglobulin binding protein from Pseudomonas maltophilia

We have demonstrated that Pseudomonas maltophilia (ATCC No. 13637) possesses an exposed, immunologically accessible protein which binds to the Fc region of several species of immunoglobulins. Whole bacteria suspensions were incubated for 18 h with purified 125I-labelled antibodies with and without added non-labelled immunoglobulins. The suspensions were centrifuged for 30 min and the pellet containing bacteria was assessed for radioactivity. Using this crude assay, the whole organism bound 125I-labelled rabbit and mouse immunoglobulins and the purified Fc portion of human IgG. All of these labelled preparations were competitively displaced by unlabelled rabbit and mouse immunoglobulins, and Fc of human IgG, as well as human immunoglobulin subclasses. The organism was sonicated to solubilize this immunoglobulin binding protein. Using this sonicated preparation, it was shown that unlabelled Fc of IgG, unlabelled mouse and rabbit immunoglobulins, all competitively displaced 125I-labelled human Fc of IgG in a dose-response manner. A partially purified protein was prepared by Sephacryl S-300 followed by Sephadex G-100 column chromatography. This preparation was incubated with 125I-Fc gamma and with the following purified unlabelled preparations: F(ab')2 of IgG, Fc of IgG, murine monoclonal IgA, IgG1, IgG2, IgG3, and IgG4. All except F(ab')2 of IgG produced dose response competitive displacement. The molecular weight, as estimated by SDS-PAGE and Western blot, was 30,000 daltons. In Western blots, Fc gamma, murine monoclonal IgA, and human immunoglobulin subclasses, all showed affinity for the immobilized protein. Human F(ab')2 fragments did not show affinity for the protein. Radioiodinated pseudomonal Ig-binding protein showed affinity for human IgG coupled to Sepharose, and was displaced by unlabelled pseudomonal Ig-binding protein. Scatchard analysis of binding showed two binding affinities: two distinct types of Ig-binding proteins were obtained, a high affinity with Kd = 1.54 x 10(-10) and a lower affinity with Kd = 2.36 x 10(-8). This immunoglobulin binding protein may be useful in immunoglobulin purification or identification.     

Binding of the two components of C2 toxin to epithelial cells and brush borders of mouse intestine.

C2 toxin elaborated by Clostridium botulinum types C and D is composed of two nonlinked protein components and has enterotoxic activity, for which the cooperation of these two components is necessary. In the present study, the binding of components I and II, the two components of C2 toxin, to isolated epithelial cells and brush borders of mouse intestine was examined. Immunofluorescence studies showed that component II, either trypsinized (T-II) or untrypsinized (UT-II), bound to the cells and the brush borders of mouse intestine, whereas component I alone did not. The binding of I was observed only when the cells and the brush borders were reacted with T-II, but not when they were reacted with UT-II. These results are consistent with the fact that the biological activities of C2 toxin are elicited by the combination of I and T-II, but not of I and UT-II. The in vitro binding of I and II to isolated brush borders of mouse intestinal cells also showed similar binding characteristics. The binding of I and II to brush borders was rapid and not temperature dependent. Ultracentrifugal analysis revealed that both I and T-II bound to microvillous membranes of the intestinal cells. The data from the present study indicate that the enterotoxic activity of C2 toxin is initiated by the binding of T-II to the microvillous membrane of intestinal cells followed by that of I, for which the site of the cell membrane is induced by the binding of T-II, but not of UT-II.     

7S immunoglobulins of a monotreme, the Echidna Tachyglossus aculeatus: two distinct isotypes which bind A protein of Staphylococcus aureus

7S immunoglobulins of a monotreme mammal, the echidna Tachyglossus aculeatus, bind to Staphylococcus aureus A protein coupled to an insoluble matrix. This binding supports the homology between the previously described echidna IgG and IgG molecules of higher mammals and provides a rapid, simple method for the isolation of this protein from echidna serum. In addition, another echidna 7S immunoglobulin became bound to protein A-Sepharose. The major protein A-binding immunoglobulin has a slow electrophoretic mobility and possesses a heavy chain comparable in mass to typical mammalian γ chains (52,000 daltons). We suggest the designation IgG2 for this immunoglobulin. The minor protein A-binding immunoglobulin is electrophoretically faster than IgG2 and can be resolved from the former by gradient elution from DEAE-Biogel. We propose the nomenclature IgG1 for this molecule although we cannot discount the possibility that it might represent another main class such as IgA. The γ1 heavy chain is distinguishable from the γ2 chain on polyacrylamide gel electrophoresis in sodium dodecylsulphate containing buffers. The γ1 chain possesses a nominal mass of 61,000 daltons. The intact molecule has an apparent mass of 177,000 Daltons and comprises two pairs of light and heavy chains.     

The half-lives of serum immunoglobulins in adult mice

We determined the half-lives of several sets of murine monoclonal antibodies spanning all immunoglobulin isotypes in the serum. The antibodies in each set possess the same V region. With this approach, the differences in half-life observed between the different isotypes are independent of the V region carried by the monoclonal antibodies and therefore must relate to each other in the same way as the half-lives of each class of serum immunoglobulins. The half-life of a monoclonal antibody of the gamma 2a isotype is identical to the average half-life of serum IgG2a as previously determined (6-8 days; P. Vieira and K. Rajewsky, Eur. J. Immunol. 1986. 16:871). Therefore, the half-lives determined with monoclonal antibodies possessing the same V region represent the half-life of the serum immunoglobulins. In this way we calculated the half-life of IgM as 2 days, IgG3 and IgG1 as 6-8 days, IgG2b has a half-life of 4-6 days. IgE has a half-life of 12 h. A polymeric form of IgA was found to be eliminated from the serum with a half-life of 17-22 h.     

Purification of immunoglobulins G by protein A/G affinity membrane chromatography

An affinity membrane grafted with protein A/G or protein A was characterized for human and mouse immunoglobulins G purification. Breakthrough curves up to ligand saturation were measured and used to study the effects of flow velocities, feed solution concentrations and protein A/G versus protein A membranes. Increased flow-rate did not decrease the amount of IgG bound to the membranes. Increased feed solution concentration allowed more IgG to bind prior to breakthrough. Kinetic parameters for immunoglobulins G sorption to immobilized protein A were measured in batch experiments. The static binding capacity was determined to be 6.6 mg ml(-1) membrane volume. Finally, this affinity membrane was used to purify IgG from cell culture supernatant. The electrophoresis of the purified IgG fractions did not show any contaminant.     

Binding of monomeric immunoglobulins by bone marrow-derived mononuclear phagocytes; its modulation by interferon-gamma

The ability of resting and activated rat bone marrow-derived mononuclear phagocytes (BMM phi) to bind monomeric rat, mouse, and human IgG was determined by means of flow cytometry. Rat IgG2b bound with high affinity (Kd approximately equal to 3 x 10(-9) M); binding was optimal at 4 degrees C and was only little affected by trypsin treatment. The other IgG bound with only low affinity (rat IgG2a, mouse and human IgG) or not at all to rat BMM phi (rat IgG1, rat IgG2c). The binding of rat IgG2b was not affected by the presence of a surplus of low-affinity binding IgG, and vice versa, indicating that high- and low-affinity IgG bind to different sites. Binding of high- and low-affinity IgG as well as expression of MHC class II molecules and of tumoricidal activity by BMM phi was markedly enhanced by rat interferon-gamma in low concentration (0.1 to 1.0 IU IFN-gamma/ml). On the other hand, heat-killed Corynebacterium parvum organisms, that were equally potent in triggering tumoricidal activity, neither enhanced the binding of IgG nor the expression of MHC class II molecules by BMM phi, suggesting that these abilities are not necessarily closely related phenomena.     

Molecular and cellular targets of anti-IgE antibodies

Immunoglobulin E (IgE) was the last of the immunoglobulins discovered. It is present in very low amounts (nano- to micro-gram per ml range) in the serum of normal healthy individuals and normal laboratory mouse strains and has a very short half-life. This contrasts with the other immunoglobulin classes, which are present in much higher concentrations (micro- to milligram per ml range) and form a substantial component of serum proteins. Immunoglobulins play a role in homeostatic mechanisms and they represent the humoral arm of defence against pathogenic organisms. Since IgE antibodies play a key role in allergic disorders, a number of approaches to inhibit IgE antibody production are currently being explored. In the recent past the use of nonanaphylactic, humanized anti-IgE antibodies became a new therapeutic strategy for allergic diseases. The therapeutic rational beyond the idea derives from the ability of the anti-IgE antibodies to bind to the same domains on the IgE molecule that interact with the high-affinity IgE receptor, thereby interfering with the binding of IgE to this receptor without cross-linking the IgE on the receptor (nonanaphylactic anti-IgE antibodies). Treatment with anti-IgE antibodies leads primarily to a decrease in serum IgE levels. As a consequence thereof, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells reducing the release of inflammatory mediator such as histamine, prostaglandins and leukotrienes. Experimental studies in mice indicate that injection of some monoclonal anti-IgE antibodies also inhibited IgE production in vivo. The biological mechanism behind this reduction remains speculative. A possible explanation may be that these antibodies can also interact with membrane bound IgE on B cells, which could interfere the IgE production.     

The five classes of immunoglobulins in normal C3H and BALB/c mice

The five major classes of mouse immunoglobulin have been purified and, by appropriate immunization of goats, monospecific antisera have been obtained. With these antisera the five classes of mouse immunoglobulin have been quantitated in the serum of normal C3H and BALB/c mice from birth to the end of the first year of life. Newborn mice of both strains have shown no IgA or IgM in their serum by the method used. Except for IgM, all immunoglobulins have shown a rapid increase a few days after birth, followed by a decline to lower levels at approximately 20–30 days of age. These changes have been observed by others and attributed to transfer of IgG1, IgG2 and IgA by colostrum and milk, but changes in the lymphoreticular apparatus are also occurring at this time which could contribute or account for some of these fluctuations. After the age of 60–80 days BALB/c mice are very often hyperglobulinaemic. This hyperglobulinaemia can be attributed to the increase in IgG1, IgM and partly IgG2b.     

Use of hybridoma immunoglobulin switch variants in the analysis of the protective properties of anti-lipopolysaccharide antibodies in Escherichia coli K1 infection

Functional properties of rat immunoglobulins obtained from hybridoma isotype switch variants were studied in vivo in a rat model for neonatal bacterial sepsis. Escherichia coli 018:K1, a common cause of human neonatal sepsis and meningitis, was injected intravenously into 6-day-old rats after incubation with 018-specific antibodies IgM, IgG1, IgG2a, IgG2b, IgG2c, IgE and IgA. The clearance of bacteria treated with saline or IgE was low, whereas monoclonal antibodies of other isotypes triggered hepatic sequestration and killing of the K1 E. coli cells. All four IgG subclasses were more efficient than IgM and IgA. Comparable results were obtained upon injecting antibodies into rats with an established fulminating bacteraemia. IgM was inactive in animals depleted of complement with cobra-venom factor (CVF), whereas IgG2b was able to trigger hepatic clearance independently of complement.     

Binding of human immunoglobulin G to protein A in encapsulated Staphylococcus aureus.

Recent studies of the mechanism of resistance to phagocytosis in encapsulated Staphylococcus aureus have suggested that the capsule is readily penetrated by high-molecular-weight proteins such as antibodies and complement components. S. aureus strains contain a cell wall protein, protein A, that reacts with the Fc portion of immunoglobulins. The binding of immunoglobulin G (IgG) to encapsulated and unencapsulated S. aureus strains has been studied to assess the penetrability of the S. aureus capsule by IgG. Encapsulated S. aureus strains M and Smith diffuse bound large amounts of human IgG which were comparable to amounts bound by the unencapsulated strains Cowan I, M variant, and Smith compact. Trypsin treatment of bacteria reduced their ability to bind IgG. Bound IgG was not removed by extensive washing of bacteria with buffer. A non-protein A-containing, coagulase-negative, encapsulated staphylococcal strain did not bind IgG. These observations suggest that IgG is binding to cell wall protein A in encapsulated S. aureus. No differences in the rates of IgG binding by encapsulated and unencapsulated S. aureus strains were observed. It is concluded that the S. aureus capsule is freely permeable to IgG. This is of importance in considerations of the mechanisms of resistance to phagocytosis and antigen masking in encapsulated microorganisms.     

Enhancement of IgG production elicited in mice by treatment with anti-CD8 antibody.

Injection of an anti-CD8 monoclonal antibody into mice was followed by an increase in the production of immunoglobulins of the IgG1, IgG2a and IgG2b, but neither the IgG3 nor the IgM isotypes. A maximal effect was observed 6 days after administration of 100 micrograms antibody. However, no modification of spleen cell proliferation was elicited by the anti-CD8 treatment. Virally induced IgG2a and IgG2b secretion remained unchanged in mice infected with lactate dehydrogenase-elevating virus that received a concomitant injection of anti-CD8 antibody, whereas an enhancement of IgG1 was observed. Simultaneous treatment with an anti-CD4 antibody abrogated the immunoglobulin secretion triggered by anti-CD8. These results suggest that immunoglobulin production in unmanipulated mice is controlled by CD8+ lymphocytes.     

Immunoglobulin Binding Specificities of the Homology Regions (Domains) of Protein A

Protein A binds immunoglobulins and it has two target structures, one in Feγ (C_H) and the other in selected V_H regions. The protein has five homology regions (domains). A, B, C, D, and E. Fc-binding and V_H-binding have been reported to be non-competitive, suggesting that different domains are responsible for the binding of the two ligands. On the other hand, all five domains have been reported to bind Fc. I studied binding of different immunoglobulins by protein A or its domain B (rBB). The results show that separate domains bind V_H and Fc. If all five domains are capable of binding Fc, the ones that bind V_h have low affinity for Fc. Furthermore, the number of Fc-binding domains varies depending on the type of the IgG being bound. Human IgG1 or lgG2 or rabbit IgG (Fc) seem to be bound by several domains (possibly four), and domain B is one of them. Mouse IgG1 or lgG2b are bound by fewer domains not including B. Murine lgG2a is also bound by fewer domains but B is one of them.     

IgEb immune complexes activate macrophages through FcgammaRIV binding

Because functional analysis of Fc receptors (FcRs) relies heavily on mouse models, the identification of another Fcgamma receptor is particularly noteworthy. We demonstrate that FcgammaRIV, identified here as the mouse ortholog of primate FcgammaRIII, required association of the FcR gamma-chain for optimal expression and function on myeloid cells; its signaling potential was also enhanced by a cytoplasmic 'YEEP' motif that was able to recruit the adaptor molecule Crk-L and phosphatidylinositol-3-OH kinase. Unexpectedly, FcgammaRIV 'preferentially' bound immunoglobulin E antibodies of the 'b' allotype (IgE(b)) as well as IgG2a and IgG2b antibodies. Ligation of FcgammaRIV by antigen-IgE(b) immune complexes promoted macrophage-mediated phagocytosis, presentation of antigen to T cells, production of proinflammatory cytokines and the late phase of cutaneous allergic reactions. IgE(b) antibody-mediated modification of macrophage responses may therefore influence mouse asthma models and strain-dependent differences in parasite susceptibility.