Transferrin-immune complex disease: a potentially overlooked gammopathy mediated by IgM and IgG.

The combination of marked hypersideremia, hypertransferrinemia, and monoclonal gammopathy of underdetermined significance (MGUS) should alert clinicians to the possible presence of an anti-transferrin immunoglobulin, an uncommon acquired disorder also defined as transferrin-immune complex disease (TICD). The authors have previously described a case of TICD with 100% transferrin saturation and liver iron overload. However, the findings in the few cases so far reported are heterogeneous, and the presence of high transferrin saturation and liver iron overload is not universal. In this article, the authors have described the identification of two additional patients with anti-transferrin monoclonal gammopathy, hypersideremia, and hypertransferrinemia, but with incomplete transferrin saturation and no hepatic iron overload. The autoantibodies were purified by using transferrin as affinity bait and characterized. One subject showed a high-titer monoclonal anti-transferrin IgM with a κ-type light chain. This finding is the first observation of IgM autoantibodies against transferrin. The other patient developed the disease after pregnancy. In this study, monoclonal antibody was an IgG mounting a κ-type light chain with altered molecular weight. These results highlight that transferrin might induce the development of a monoclonal immune response of different classes and specificity. The identification, in a single hematologic center, of three different subjects with anti-transferrin monoclonal gammopathy suggests that the disease probably represents a still underdiagnosed condition. From a clinical standpoint, these patients must be followed up both as MGUS and as hemochromatosis.

Artiodactyl IgD: the missing link.

IgD has been suggested to be a recently developed Ig class, only present in rodents and primates. However, in this paper the cow, sheep, and pig Ig delta genes have been identified and shown to be transcriptionally active. The deduced amino acid sequences from their cDNAs show that artiodactyl IgD H chains are structurally similar to human IgD, where the cow, sheep, and pig IgD H chain constant regions all contain three domains and a hinge region, sharing homologies of 43.6, 44, and 46.8% with their human counterpart, respectively. According to a phylogenetic analysis, the Cdelta gene appears to have been duplicated from the Cmu gene >300 million yr ago. The ruminant mu CH1 exon and its upstream region was again duplicated before the speciation of the cow and sheep, approximately 20 million yr ago, inserted upstream of the delta gene hinge regions, and later modified by gene conversion. A short Sdelta (switch delta) sequence resulting from the second duplication, is located immediately upstream of the bovine Cdelta gene and directs regular mu-delta class switch recombination in the cow. The presence of Cdelta genes in artiodactyls, possibly in most mammals, suggests that IgD may have some as yet unknown biological properties, distinct from those of IgM, conferring a survival advantage.

Deletion of the DQ52 element within the Ig heavy chain locus leads to a selective reduction in VDJ recombination and altered D gene usage.

The process of V(D)J recombination that leads to the assembly of Ig gene segments is tightly controlled during B cell differentiation. Two germline transcripts, one of which (mu(0)) originates from the promoter region of DQ52, may control the accessibility of the heavy chain locus. Here, we present the analysis of a mouse line in which the DQ52 gene together with its regulatory sequences is deleted by a Cre/loxP-based strategy. In F(1) (DQ52(+/-)) mice, the use of the JH3 and JH4 elements in DJ or VDJ junctions of the DQ52(-) allele was strongly reduced in both the bone marrow pre-B and spleen cells, while the JH1 and JH2 elements were used with normal frequencies. In addition, IgM(+) B cells of bone marrow and spleen used the DQ52(-) allele less frequently. On DJ joints of the DQ52(-) allele, there was 2 times less processing of JH3 ends, which resulted in clearly increased addition of P nucleotides. Although the use of D elements in DJ joints was quite similar, an altered D repertoire was found in VDJ joints of the DQ52(-) allele. In splenic B cells of the DQ52(-/-) mouse the amino acid distribution of the CDR3 was skewed, probably to compensate for the altered processing of JH3 ends. Thus, we have shown an interesting selective effect of the DQ52 region on controlling accessibility to 3' JH elements on the Ig locus, which also seems to influence the processing of DJ joints. We propose a model in which the DQ52 promoter region enhances the induction of secondary DJ rearrangements.

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials.

cDNAs encoding IgM heavy chain constant region (Cmu) were isolated from two metatherians (marsupials)--the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cmu in both species. The domain size and structure of the marsupial Cmu sequences were compared with other Cmu sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.

Switch recombination in a transfected plasmid occurs preferentially in a B cell line that undergoes switch recombination of its chromosomal Ig heavy chain genes.

Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e. g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29 mu), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of S mu-S alpha recombination in the plasmid. Recombination of the plasmid in I.29 mu cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29 mu cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.

Expressed immunoglobulin repertoire of LPS-stimulated splenocytes of unimmunized mice as studied by two-dimensional gel electrophoresis.

The repertoire of isolated immunoglobulin polypeptide chains synthesized by LPS-stimulated splenic B cells from unimmunized 6 weeks old mice was studied by two-dimensional gel electrophoresis. These B cells formed mainly mu heavy chains, while only a small amount of gamma chains was detected on two-dimensional electrophoregrams. The number and character of spots corresponding to each class and type of H and L chains were analyzed. Most of the detected 52 spots, which corresponded to L chains, were well resolved with clearly defined round boundaries. Six of them belonged to two isotypes of lambda chains and the rest to the kappa chain. About 25 clusters corresponded to mu chains. They had different appearance from those of L chains and their characteristic elliptic form with prolonged vertical axes indicated the presence of several H chain variants of slightly different length (due probably to the length variations of CDR3 and carbohydrate heterogeneity) in each cluster. The limited number of spots both of H and L chains is explained as being due to restrictions in the expressed repertoire of preimmune splenic B cells, which have no somatic mutations in the immunoglobulin genes. The concept of macrorepertoire (referring to the relatively small number of detected molecular species) and microrepertoire (describing the mutationally altered molecules) is introduced.

Resource competition as a mechanism for B cell homeostasis.

Cellular competition for survival signals offers a cogent and appealing mechanism for the maintenance of cellular homeostasis [Raff, M. C. (1992) Nature (London) 356, 397-400]. We present a theoretical and experimental investigation of the role of competition for resources in the regulation of peripheral B cell numbers. We use formal ecological competition theory, mathematical models of interspecific competition, and competitive repopulation experiments to show that B cells must compete to persist in the periphery and that antigen forms a part of the resources over which B cells compete.

Molecular components and assembly of mu.surrogate light chain complexes in pre-B cell lines.

We describe the molecular components, subunit assembly, and cell surface expression of mu-chain complexes in mu+kappa- pre-B cell lines as revealed by two-dimensional gel electrophoresis. The mu-chain complexes of these cell lines contain several previously unreported components, p42(6.4), p39(6.7), p18(8.6), and p14(7.0), in addition to lambda 5, VpreB1, VpreB3 (formerly named 8HS20), MB-1(Ig-alpha), and B29(Ig-beta). These new components are not detected in mu+kappa+ immature B cell lines. The mu-chain associates with lambda 5, VpreB3, and p56(5.0) at an early phase of assembly, preceding the association of other molecules. mu-Associated VpreB3 decreased during assembly as the association of VpreB1 became dominant, suggesting that the change in the ratio of these two VL-like surrogate light chains is involved in the mechanism of assembly. Lambda 5, VpreB1, p56(5.0), p32(5.0), p36(5.5), and p14(7.0) were shown to be expressed on the cell surface in association with mu-chain. The association of the other molecules with mu-chain is most likely restricted to the intracellular compartment. An interaction between VpreB1 and VpreB3 was also suggested. These findings might be important for understanding the function of mu-chain complexes in pre-B cells. A possible signaling mechanism of mu/surrogate light chain complexes is discussed.

Two types of mu chain complexes are expressed during differentiation from pre-B to mature B cells.

Immunoglobulin mu chains synthesized in murine pre-B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre-B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross-linking of micron chains on the surface of pre-B cells resulted in an increase in intracellular free Ca2+, indicating that the micron chain complex on the surface of pre-B cell lines acted as a signal transduction molecule. However, the receptor cross-linkage of pre-B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre-B to mature B cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.

The DJH complex remains active in recombination to VH segments after the loss of mu-chain expression in mu-positive pre-B cells.

AT11-2 is an Abelson virus-transformed B precursor cell line which is capable of differentiating Ig- from mu+ cells via functional recombination of VH segments to preexisting DJH complexes. We describe here that after a mu+ subclone (VDJ+/DJ) generated from Ig- AT11-2 (DJ/DJ) cells by in vitro functional VH to DJH recombination subsequently lost mu-chain expression either by the recombination of a pseudo VH segment to the VHDJH+ allele or by the deletion of VHDJH+ allele, a novel productive joining of VH segments to the preexisting DJH complex occurred. These results indicated that VH to VHDJH rearrangement was not suppressed in mu-chain producing cells and that the DJH complexes still remained active in the recombination to VH segments after the loss of mu-chain expression. Our results may also suggest that VH to DJH rearrangement, but not VH to VHDJH rearrangement, is suppressed in mu-chain producing cells to maintain allelic exclusion. Our cell differentiation system should continue to be valuable for elucidating the mechanism of suppression and associated implications regarding allelic exclusion.

Degradation of a monoclonal anti-mu chain antibody in a human surface IgM-positive B cell line starts in prelysosomal vesicle.

The surface IgM-mediated endocytosis and intracellular transport of an anti-F(c mu)5 mAb was studied by using subcellular fractionation in sucrose gradients. The results of such experiments showed that antibody was initially endocytosed in vesicles of low density, and later transferred to a presumably lysosomal compartment of higher density. SDS-PAGE analysis of gradient fractions showed that high Mr degradation fragments of the endocytosed antibody were formed in the low density vesicles before terminal degradation could be recorded. The partial degradation of the antibody was not blocked by low temperature or enzyme inhibitors, such as leupeptin and benzyloxycarbonyl-phenylalanylalanine-diazomyethyl-ketone, all of which severely retarded terminal degradation. The data also suggested that the recycling of partially degraded antibody to the cell surface employed a pool of such low density prelysosomal vesicles.

Anti-Listeria monocytogenes immunity in mu-suppressed mice: a comparison of treatment with conventional hyperimmune rabbit anti-mouse IgM and affinity-purified, monoclonal rat anti-mouse IgM.

The capacity of anti-IgM treated, B-cell-depleted mice to control infection by Listeria monocytogenes was evaluated. Suppression was achieved with a hyperimmune rabbit anti-mouse-IgM antiserum (IRS), with affinity-purified IRS (IRP), or with an affinity-purified, monoclonal, rat anti-mouse-IgM antibody (LO-MM-9). B-cell depletion in specifically treated mice was judged to be complete by the following criteria: absence of significant response to a B-cell mitogen lipopolysaccharide, absence of B-cells with detectable IgM or kappa light chain on their surface, absence of detectable IgM, and presence of free anti-IgM antibodies in serum. BALB/c mice, conventionally treated from birth with IRS, had an increased capacity to clear L. monocytogenes from the blood during the first 5 min after intravenous infection. Furthermore, control of infection seemed to be enhanced during the first 24 h but was found to be impaired when assessed 3 and 4 days after initiation of infection. These effects were, however, not IRS specific, because control mice treated with normal rabbit serum behaved comparably. Mortality caused by 2 x 10(3) L. monocytogenes injected intraperitoneally into BALB/c mice susceptible to L. monocytogenes was increased more in NRS-than in IRS-treated mice when both were compared with untreated control mice. Therefore, chronic injection of IRS or NRS seemed to disturb anti-L. monocytogenes immunity, rendering an evaluation of the role of antibodies impossible.(ABSTRACT TRUNCATED AT 250 WORDS)

Formation of disulphide-linked mu 2 omega 2 tetramers in pre-B cells by the 18K omega-immunoglobulin light chain.

Pre-B cells are precursors of B lymphocytes that contain intracellular heavy-chain protein (mu) and are either yet to rearrange their light-chain genes or are in the process of doing so. These cells have traditionally been considered to contain intracellular mu-chain with no associated light chain. We demonstrate here that pre-B lymphoid lines synthesize a protein of relative molecular mass (Mr) 18,000 (18K), which we term omega, which forms disulphide-linked mu 2 omega 2 tetramers. This protein could be immunoprecipitated with mu-chain from pre-B lines, but not from T-cell and fibroblast lines that express transfected mu-genes, nor from a pre-B line that synthesizes a D mu-protein (which lacks a V domain). We view the omega-chain as being a pre-B specific surrogate light chain that may be essential for the important regulatory function that the mu-protein is believed to have at this stage of differentiation.

Preparation of anti-bovine and porcine mu chain sera.

Absorption of anti-L chain antibodies contained in anti-IgM sera immunized with purified bovine and porcine IgM was undertaken by affinity chromatography on a column of Sepharose 4B, coupled with the normal follicular fluids of the respective species as an immunosorbent. Anti-L chain antibodies in both anti-IgM sera were completely absorbed without incurring reduction of the antibody titer, and anti-bovine and anti-porcine mu chain sera were prepared.

Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised in E. coli.

Genes for a murine mu heavy chain and a lambda light chain immunoglobulin have been inserted into bacterial expression plasmids containing the Escherichia coli trp promoter and ribosome binding site. Induction of transcription from the trp promoter results in accumulation of both light and heavy chain polypeptides in appropriate host strains. Both proteins were found as insoluble products. Following extraction and purification of the immunoglobulin containing fractions, antigen binding activity was recovered. The activity demonstrates essentially the same properties as the antibody from the hybridoma from which the genes were cloned.

Signet-ring cell variant of large cell lymphoma.

A diffuse, large cell lymphoma of palatine tonsil was found to contain a considerable number of enlarged tumor cells with prominent, hyaline, Russell body-type cytoplasmic inclusions displacing the nucleus peripherally and, thus, the morphologic features of signet-ring cell lymphoma. Immunoperoxidase staining revealed that the contents of the signet-ring cells were strongly positive for mu heavy chains and kappa light chains. Ultrastructurally, Russell body-type inclusions consisted of multiple, angulated, electron-dense crystalloids enclosed within expanded segments of rough endoplasmic reticulum.

A peptide difference between the mu-chains from cell-associated and secreted IgM of the BCL1 tumor.

The murine B cell tumor, BCL1, bears monomeric IgM lambda on its surface. After stimulation in vitro with LPS, the cells secrete pentameric IgM lambda. Comparison of mu-chains from radiolabeled intracellular, surface, and secreted IgM indicates that mu-chains from the three sites have different apparent m.w. Since the observed differences are analogous to those reported for normal murine lymphoid cells, the BCL1 cells were used for determining the structural basis for the differences in m.w. of mu-chains from the above sites. Comparative peptide analysis was performed on mu-chains from cell associated and secreted IgM. Approximately 25 peptides were identified after digestion with chymotrypsin and trypsin and analysis of peptides by cation exchange chromatography. All peptides co-eluted with the exception of a single extra peptide derived from the Fc portion of the secreted IgM. The same peptide was observed in a similar analysis using mu-chains from IgM secreted by normal splenocytes.