Expression, distribution and specificity of Fc receptors for IgM on murine B cells

Subpopulations of normal adult murine splenic B cells and a panel of murine B cell tumors were examined for their ability to bind murine IgM specifically. By using two-color flow cytometric analyses, we have demonstrated that 90 to 95% of surface (s)IgD+ B cells express surface membrane receptors for IgM (Fc mu R). The binding of pentameric murine IgM to splenocyte Fc mu R was IgM-specific since it was totally inhibited by other polymeric IgM proteins, but not by Ig of other H chain classes or by mAb specific for the murine IgG or IgE FcR. Binding of IgM to splenic cells was saturable. Fc mu R were co-expressed with the Fc gamma R as well as the Fc epsilon R on the majority of splenic B cells. Minor populations of splenic mononuclear cells expressed only an Fc mu R, Fc gamma R or Fc epsilon R. In a survey of B tumor cell lines representing different stages of B cell development, we observed that the Fc mu R was expressed on pre-B cell lines and that Fc mu R detection was maximal on immature B cell lines that expressed sIgM and low amounts of sIgD and Ia. Fc mu R were not detected on cell lines that had switched from sIgM to the expression of another sIg, or on plasmacytomas and hybridomas. The studies with normal splenocytes establish that the majority of sIgD+ B lymphocytes in adult BALB/c mice express surface membrane receptors that specifically bind IgM. The studies with B lineage tumor cells suggest that the expression of Fc mu R on B cells is developmentally regulated and that the pattern of expression exhibited by Fc mu R during B cell ontogeny differs from the patterns that have been previously found for IgG and IgE FcR. These observations raise the possibility that Fc mu R might have a functional significance in some aspect of B cell maturation and activation. By using a family of IgM H chain constant region domain deletional mutants, we have further demonstrated that, like the T cell Fc mu R, the B cell Fc mu R also requires a C mu 3 domain for binding to occur, raising the possibility that the T and B cell Fc mu R in mice may be structurally related molecules.     

C-reactive protein binding to murine leukocytes requires Fc gamma receptors

Human C-reactive protein (CRP) is an acute phase protein that binds to receptors on human and mouse leukocytes. We have recently determined that the high and low affinity receptors for CRP on human leukocytes are Fc gamma RIIa and Fc gamma RI, respectively. Previous work by others suggested that CRP receptors on mouse macrophages are distinct from Fc gamma R. We have taken advantage of the availability of mice deficient in one or more Fc gamma R to reexamine the role of Fc gamma R in CRP binding to mouse leukocytes. Three strains of Fc gamma R-deficient mice were examined: gamma-chain-deficient mice that lack Fc gamma RI and Fc gamma RIII, Fc gamma RII-deficient mice, and mice deficient in both gamma-chain and Fc gamma RII that lack all Fc gamma R. No binding of CRP was detected to leukocytes from double-deficient mice, indicating that Fc gamma R are required for CRP binding. CRP binding to leukocytes from gamma-chain-deficient and Fc gamma RII-deficient mice was reduced compared with binding to leukocytes from wild-type mice. Further analysis of CRP binding to macrophages, neutrophils, and lymphocytes provides direct evidence that Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RI are the receptors for CRP on mouse leukocytes. These findings may have important implications in understanding the physiological function of CRP.     

IgA-induced avidity maturation of IgA Fc receptors on murine T lymphocytes

The analysis of 30 well characterized murine T lymphocyte populations using a cytofluorometric IgA binding assay has identified many populations that are constitutive and/or inducible for IgA receptor expression, and has identified two distinct mechanisms by which IgA up-regulates the IgA-binding properties of murine T cells. Studies with lymphomas, hybridomas, Ag-specific clones and activated normal splenic T cells identified many examples of CD4 and CD8 lineage cells that constitutively expressed IgA receptors. T cell populations that constitutively expressed IgA receptors exhibited enhanced IgA binding after incubation with oligomeric IgA for 18 h. The IgA-induced up-regulation of IgA binding resulted from two distinct processes: 1) an increase in the number of surface membrane IgA binding sites and 2) an increase in the avidity of IgA binding without a change in the number of binding sites. The IgA-induced avidity increase was reflected by a 5- to 10-fold decrease in the apparent Kd. Depending on the T cell population examined the enhanced binding of IgA involved one or both of these mechanisms. T cell populations that did not constitutively express IgA receptors failed to bind IgA after prolonged incubation with oligomeric IgA suggesting that if such cells can express IgA receptors they require other signals to induce their expression. Consistent with this possibility is the finding that resting splenic T cells did not bind IgA but their activation with Con A or mAb anti-T3 resulted in high level expression of IgA receptors. These studies have identified multiple distinct mechanisms that alter the IgA-binding properties of murine T cells and are discussed in terms of their possible physiologic significance.     

Divergent appearance of complement receptors and Fc receptors on T cells in a murine mammary tumor system.

The emergence of novel T cells during mammary tumorigenesis has been previously described. T cells with surface markers usually associated with B cells, i.e. complement receptors (CR), appear in the spleens from tumor bearing mice. We now report on the appearance of Fc receptor (FcR) positive T cells in the spleens from the same animals. The kinetics of appearance of the two kinds of cells are similar.Based on evidence from double and triple label assays, it was concluded that FcR and CR are not coexpressed on the same T cell and that the two kinds of T cells which emerge do so in an independent fashion. Furthermore, they appear to represent a branch in the differentiation process influenced by tumor growth. The development of CR⁺ T cells represents an irreversible process as evidenced by the lack of change in the cells' representation following surgical procedures. In contrast the development of FcR⁺ T cells appears to be quite flexible in nature since mere surgical trauma as well as tumor mass removal can effect a decrease in the proportion of such cells.     

In vitro and in vivo activation of murine gamma/delta T cells induces the expression of IgA, IgM, and IgG Fc receptors.

The present studies examined resting and activated murine gamma/delta T lymphocytes, in vitro and in vivo, for surface expression of FcR. Polyclonal gamma/delta TCR+ lymphocytes selectively grown from the spleen and intestine of normal mice did not express FcR when the cells were in a resting state, but when cells were activated with anti-CD3 antibody virtually all of the splenic gamma/delta lymphocytes and a large subpopulation of the intestinal gamma/delta lymphocytes expressed IgA and IgM FcR. This was confirmed by using transgenic mice. Resting gamma/delta TCR+ lymphocytes from the spleen, thymus, lymph node, and blood of gamma/delta TCR transgenic mice did not express FcR for any of the five major classes of Ig H chains. Activation of the gamma/delta TCR+ cells via the CD3/TCR complex induced high levels of IgM and IgA FcR and low levels of IgG FcR. Finally, in hepatic granulomas of schistosome-infected mice, activated gamma/delta TCR+ cells are present and express high levels of IgA and IgM FcR and low levels of IgG FcR. These investigations establish that transition of gamma/delta TCR+ lymphocytes from a resting to an activated state (triggered via the T3Ti TCR complex) is accompanied by the induction of surface membrane receptors specific for Ig H chain isotypes. The activation-linked expression of FcR on gamma/delta TCR+ lymphocytes provides potential mechanisms for coupling the functional activities of gamma/delta T lymphocytes with immune mechanisms that involve Ig molecules, such as antibody-dependent cellular cytotoxicity.     

Chimeric Fc receptors identify functional domains of the murine high affinity receptor for IgG.

Chimeric Fc gamma R have been generated between the mouse high affinity receptor for IgG (Fc gamma RI) and the low affinity receptor for IgG (Fc gamma RII) by exchanging the first two domains of the three-domain extracellular structure of Fc gamma RI with the homologous two-domain extracellular structure of Fc gamma RII. Studies of the affinity and specificity of binding of mouse Ig classes to these receptors defined functional regions of Fc gamma RI and showed some surprising results. After removal of the third extracellular domain of Fc gamma RI, the remaining two domains (domains 1 and 2) retained the capacity to bind Ig in the form of immune complexes, however, they bound monomeric IgG2a with a reduced affinity. Surprisingly, these two domains in the absence of the third domain bound not only IgG2a but also IgG1 and IgG2b, i.e., the third domain of Fc gamma RI suppresses the intrinsic capacity of the first two domains to act as a low affinity Fc gamma RII-like molecule. Linking the third extracellular domain of Fc gamma RI to the two extracellular domains of Fc gamma RII resulted in a receptor that retained the specificity and affinity of Fc gamma RII. Thus, the removal of domain 3 from Fc gamma RI resulted in the conversion of Fc gamma RI to an "Fc gamma RII-like" receptor. These findings indicate that domains 1 and 2 of Fc gamma RI form an Ig-binding motif, and although domain 3 is not essential for Fc binding by Fc gamma RI, it plays a crucial role in determining the specific high affinity interaction of Fc gamma RI with IgG2a.     

Fc receptors on human T lymphocytes. II. Cytotoxic capabilities of human T gamma, T mu, B, and L cells.

Subpopulations of human peripheral blood lymphocytes were prepared by rosetting techniques employing neuraminidase-treated sheep erythrocytes (SRBC_n), sheep erythrocytes coated with IgM and murine complement (EAC′), and bovine erythrocytes coated with IgG and IgM. The isolated subpopulations were tested in assays of natural cytotoxicity (NC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). B cells (SRBC_n?, EAC′+) did not mediate cytotoxicity. L cells (SRBC_n?, EAC′?) mediated NC and ADCC but not MICC. T cells (SRBC_n+) mediated NC, ADCC, and MICC. Separation of T cells into Fc-IgG (Tγ) and Fc-IgM (Tμ) subsets revealed that Tγ cells mediated NC, ADCC, and MICC while Tμ cells mediated only MICC. Thus MICC but not NC or ADCC was solely T-cell mediated. Tγ and L cells were functionally distinguishable in that Tγ cells but not L cells mediated MICC. Tγ cells and Tμ cells differed with regard to NC and ADCC effector function while both subsets mediated MICC.     

Transmembrane domains in the functions of Fc receptors

In the present study, we use a novel method, PHDhtm, to predict the exact locations and extents of the transmembrane (TM) domains of multisubunit immunoglobulin Fc-receptors. Whereas most previous studies have used single residue hydrophobicity plots for characterizing of these domains, PHDhtm utilizes a system of neural networks and the evolutionary information contained in multiple alignments of related sequences to predict the above. Present PHDhtm application predicts TM domains of immunoglobulin Fc-receptors that in many cases differ significantly from those derived by using earlier methods. Comparisons of helical wheel projections of the presently derived TM domains from PHDhtm with those produced earlier reveal different hydrophobic moments as well as hydrophobic and hydrophilic surfaces. These differences probably alter the character of subunit association within the receptor complexes. This new algorithm can also be used for other membrane protein complexes and may advance both understanding the principles underlying such complexes formation and design of peptides that can interfere with such TM domain association so as to modulate specific cellular responses.     

Detection of soluble immune complexes by their binding to Fc receptors on mastocytoma cells

Adenosine kinase activity in in vitro human peripheral blood monocyte and human pulmonary alveolar macrophage cultures undergoes significant increases, 3- to 10-fold, in both total and specific activity during 14 days culture. Increased activity in monocyte cultures was not detected during the first 3 days of culture. Adenosine kinase activity in both mononuclear phagocyte cell cultures had a pH optimum at 6.0 and activity was dependent on the concentration of ATP and magnesium; 5 mM ATP and 2.5 mM MgCl were optimal. Increased concentrations of ATP or magnesium were inhibitory. Both dATP and GTP served as phosphate donors in the absence of ATP; in contrast, pyrimidine triphosphates were poor donors. Enzyme activity was inhibited by 1 μM p-chloromercuribenzoate and substrate inhibition by excess adenosine was observed in 2-week pulmonary alveolar macrophage cultures but not in freshly isolated cells. The role of increased adenosine kinase activity in in vitro monocyte-macrophage differentiation is considered.     

Structural studies of T lymphocyte Fc receptors. Association of Gc protein with IgG binding to Fc gamma

To obtain further information concerning the structure of Fc IgG-binding sites on human peripheral blood lymphocytes, enriched T-cells were surface-radioiodinated and treated with nonionic detergent, and the soluble supernatant was submitted to affinity chromatography selecting for components binding complexed IgG. Analysis of eluted material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis demonstrated the major proteins to be of Mr 56,000, pI 4.8-5.1 and Mr 60,000, pI 5.0-5.6, and these were radiolabeled, indicating an origin in part from the T-cell membrane. While the Mr 56,000 band gave positive reactions upon transblotting with antisera to Gc protein, the identity of the Mr 60,000 protein remains unknown. Three other components were detected, although with less consistency; Mr 78,000, 42,000, and 20,000-25,000, respectively. Immunocytochemical experiments showed that less than 5% freshly isolated native T-cells were positive with antiserum to human Gc, but, after IgG antibody-coated erythrocyte rosetting, the number of positive cells increased to 15-25%, in close agreement with the percentage of IgG antibody-coated erythrocyte rosette-positive T-cells. These findings therefore indicate that, in addition to interaction with components of Mr 60,000, 42,000, and 20,000-25,000, complexed IgG binding to Fc gamma of human peripheral blood T-cells also becomes spatially associated with Gc protein.     

Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI.     

Molecular characteristics of IgA and IgM Fc binding to the Fcalpha/muR

Fcalpha/mu receptor (Fcalpha/muR), a novel Fc receptor for IgA and IgM, is a type I transmembrane protein with an immunoglobulin (Ig)-like domain in the extracellular portion. Although IgA and IgM bind to Fcalpha/muR, the molecular and structural characteristics of the ligand-receptor interactions have been undetermined. Here, we developed twelve monoclonal antibodies (mAbs) against murine Fcalpha/muR by immunizing mice deficient in Fcalpha/muR gene. Eight mAbs totally or partially blocked IgA and IgM bindings to Fcalpha/muR. These blocking mAbs bound to a peptide derived from the Ig-like domain of murine Fcalpha/muR, which is conserved not only in human and rat Fcalpha/muR but also in polymeric Ig receptor (poly-IgR), another Fc receptor for IgA and IgM. These results suggest that IgA and IgM bind to an epitope in the conserved amino acids in the Ig-like domain of Fcalpha/muR as well as poly-IgR.     

Fc receptors of endothelial cells of cardiac valves: comparison of IgG Fc binding activity of these receptors and of Fc receptors of group A streptococci

Normal human and rabbit sera, as well as IgG isolated from them, have proved to be capable of reacting with the cells of the valve endothelium of the human and bovine heart. As shown in this study, these reactions are linked with the presence of Fc receptors on the epithelial cells. This is confirmed by the positive reactions of the endothelial cells with the Fc fragments of IgG, as well as with pure antibodies to egg albumin and to group A streptococcal polysaccharide and their complexes. As revealed in this study, Fc receptors on endothelial cells and staphylococcal Fc receptors bind with the definite fraction of normal human serum IgG with, probably, more pronounced cytophil properties. This fraction is not linked with IgG subclasses. The suggestion may be made that the presence of IgG Fc binding activity in group A streptococci, coinciding with the binding activity of Fc receptors in some cells of the human body, is probably of importance for pathogenic streptococci, facilitating their successful invasion.     

IgM complex receptors on subpopulations of murine lymphocytes.

Murine lymphocytes from spleen, lymph node, and thymus were examined for IgM complex receptors. Lymphocytes from all three organs were found to bind SRBC sensitized with IgM from various sources including: primary anti-SRBC serum, murine and rabbit anti-Escherichia coli LPS sera, and a murine IgM myeloma (MOPC 104E). Rosette formation by lymphocytes with IgM-sensitized SRBC was inhibited by soluble antigen-IgM complexes but not by IgM or antigen alone. Rosette formation was also inhibited by human IgM (Fc)5mu but not by Fab mu. Antiserum and complement treatment of the cells and subsequent recovery of the viable cells by trypsinization, filtration, and washing revealed the IgM rosette-forming cell (RFC) in the thymus to be a T cell. Spleen on the other hand was found to contain both B and T cells capable of binding IgM sensitized SRBC. Removal of both B and T cells from spleen cell suspensions eliminated all IgM RFC. The IgM complex receptor was found to be trypsin insensitive. Anti-Ig column fractionation enriched IgM RFC in spleen and lymph node suspensions passed through the columns, whereas cells bearing surface Ig, IgG complex receptors, and C3 receptors were retained in the columns.     

Metabolic and morphologic effects of colchicine on human T-lymphocyte expression of Fc mu and Fc gamma receptors

The influence of colchicine on human T-cell Fc mu- and Fc gamma-receptor expression during culture was studied utilizing a rosette technique with bovine erythrocytes coated with IgM (EOx-IgM) or IgG (EOx-IgG). Treatment of T cells with greater than or equal to 10(-6) M concentrations of colchicine induced in these cells progressive loss of microtubules and surface microvilli, inhibited their Fc mu-, but not Fc gamma-receptor expression during culture, and increased their cyclic AMP levels. However, similar treatment of cells with lumicolchicine, a photoinactivated isomer, identically inhibited the T-cell Fc mu-receptor expression as well, without inducing loss of microtubules or microvilli or raising cyclic AMP levels in them. A direct influence on T-cell protein synthesis by either colchicine or lumicolchicine is likely, as greater than or equal to 10(-6) M concentrations of alkaloid identically inhibited [3H]leucine incorporation and Fc mu-receptor expression by T cells without inhibiting their alpha-methyl isobutyric acid transport. No impairment of optimal EOx-IgM rosette formation occurred in control T lymphocytes cultured for 24 hr and then treated with colchicine, which suggests that its effects did not directly influence the receptor-ligand interaction itself. These findings suggest colchicine has several sites of action on T cells, dependent and independent of microtubular depolymerization, which may be responsible for alterations of T-lymphocyte cellular metabolism and function.     

Sequence-dependent contribution of distal binding domains to CAP protein-DNA binding affinity.

We report measurements of the relative binding affinity of CAP for DNA sequences which have been systematically mutated in the region flanking the consensus binding site. Our experiments focus on the locus one helical turn from the dyad axis where DNA bending toward the minor groove is induced upon C-AP binding. The binding free energy and extent of bending are moderately well correlated for the set of 56 sequences. Changes in binding affinity spanning a factor of about 50 could be accounted for by additive contributions of dinucleotides; with a few exceptions, the relative ranking of dinucleotide contributions to binding and bending are similar. We conclude that dinucleotides are the smallest independent unit required for quantitative interpretation of CAP-induced DNA bending and binding in the distal domains of the CAP consensus binding site. The imperfect correlation between binding strength and extent of bending implies that sequence changes affect protein binding strength not only by altering the DNA deformation energy required to form the complex, but also by affecting directly the free energy of interaction between protein and DNA.     

Characterization of the Fc receptors of the murine leukemia L1210

A glycoprotein extract prepared from the plasma membranes of L1210 cells was passed over columns of Sepharose 4B to which either heat-aggregated human IgG or F(ab′)₂ fragments had been coupled. The intact IgG column bound 35.7% of the applied counts, whereas the F(ab′)₂ columns bound 2.8%. The bound glycoproteins were eluted with citrate buffer (pH 3.2) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three peaks with apparent molecular weights of 65,000, 45,000, and 28,000 daltons were identified and purified by electroelution from polyacrylamide gels. The isolated proteins were able to bind to the same subclasses of mouse IgG myeloma proteins as the intact L1210 cells, indicating that these molecules are related to L1210 surface Fc receptors. Amino acid analyses of the 3 proteins were markedly similar suggesting that the observed molecular heterogeneity might be due to carbohydrate differences. Neuraminidase digestion of the isolated proteins resulted in mobility shifts on polyacrylamide gel electrophoresis which were consistent with the interpretation that either the isolated proteins have considerably different sialic acid contents, or that removal of the sialic acid results in disaggregation of an Fc receptor molecule.