Control of immunoglobulin secretion in the murine plasmacytoma line MOPC 315.

Cells of the 315LV-1 (derived from NP1) variant line of MOPC 315 contain approximately 1% the normal intracellular level of the heavy (alpha) chain of IgA and no detectable light (lambda2) chain. The synthesis rate of alpha-chain in the variant, however, is similar to that in cells of the parent line. Moreover the relative amount of translatable alpha-chain mRNA that can be extracted from 315LV-1 cells is about the same as for parental cells. No light-chain synthesis can be detected either in vivo or in vitro in a wheat germ cell-free system. The 315LV-1 heavy chain synthesized in vivo or in vitro has slightly greater electrophoretic mobility than normal H chain and turns over rapidly intracellularly. The variant fails to secrete any of its heavy chain, despite the fact that its H chain mRNA is bound to membranes, as one would expect for a secretory protein message. Fusion of 315LV-1 cells with cells of a kappa-producing MPC 11 variant line leads to stabilization of the intracellular H chain and also to full recovery of secretion of the H chain as an H2L2 molecule.

Counterselection against D mu is mediated through immunoglobulin (Ig)alpha-Igbeta.

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (D mu). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Ig alpha-Ig beta, whereas the mechanism for counterselection against D mu has not been determined. We have examined the role of the Ig alpha-Ig beta signal transducers in counterselection against D mu using mice that lack Ig beta. We found that D mu expression is not selected against in developing B cells in Ig beta mutant mice. Thus, the molecular mechanism for counterselection against D mu in pre-B cells resembles positive selection in that it requires interaction between mD mu and Ig alpha-Ig beta.

In vivo growth advantage of the MOPC-315 mouse myeloma over its immunoselected variants.

Myeloma cells of the "wild type" that produce complete immunoglobulin molecules and those of the more usual variant type that display only one kind of chain [either light (L) or heavy (H)] were cocultivated ip and sc in syngeneic BALB/c mice. With each of six deliberately selected variants, a progressive increase in the proportion of wild-type cells was observed; the rate of change suggested that these variants had an approximately 10% slower growth rate than that of the wild-type tumor. In contrast, a variant that arose spontaneously overgrew the wild-type cells. The results may account for a) the stable capacity of most wild-type tumors to produce complete immunoglobulin molecules (L- plus H-chains) over many years, even though they frequently generate variant cells that produce only L- or only H-chains; and b) the occasional spontaneous change of myeloma cell populations from predominantly wild-type to variant cells.

Measurement of messenger RNA encoding the alpha-chain, polymeric immunoglobulin receptor, and J-chain in duodenal mucosa from dogs with and without chronic diarrhea by use of quantitative real-time reverse transcription-polymerase chain reaction assays.

OBJECTIVE: To examine the difference in expression of messenger RNA (mRNA) transcripts for polymeric immunoglobulin receptor (plgR), alpha-chain, and J-chain determined by use of quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) assays in duodenal biopsy specimens obtained from dogs with and without chronic diarrhea. SAMPLE POPULATION: Biopsy specimens of the proximal portion of the duodenum were obtained endoscopically from 39 dogs evaluated because of chronic diarrhea (12 German Shepherd Dogs and 27 non-German Shepherd Dog breeds); specimens were also obtained from a control group of 7 dogs evaluated because of other gastrointestinal tract diseases and 2 dogs that were euthanatized as a result of nongastrointestinal tract disease. PROCEDURE: Dogs were anesthetized, and multiple mucosal biopsy specimens were obtained endoscopically at the level of the caudal duodenal flexure by use of biopsy forceps; in 2 control dogs, samples were obtained from the descending duodenum within 5 minutes of euthanasia. One-step QRT-PCR was used to quantify the level of expression of transcripts for the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase, plgR, alpha-chain, and J-chain in duodenal mucosal tissue. RESULTS: There was no significant difference in the level of expression of any transcript among non-German Shepherd Dog breeds without diarrhea (control group), non-German Shepherd Dog breeds with chronic diarrhea, and German Shepherd Dogs with chronic diarrhea. Conclusions and Clinical Relevance-Results indicated that the susceptibility of German Shepherd Dogs to chronic diarrhea is not a result of simple failure of transcription of the key genes that encode molecules involved in mucosal IgA secretion.

IgA polymorphism in mice: individual light chains can pair covalently with some alpha heavy chains and non-covalently with others.

The molecular basis for the two different forms of IgA in mice, distinguished by the covalent or non-covalent association of light (L) and heavy (H) chains, is unknown. In this communication, we show, using somatic cell hybridization to construct cells producing new combinations of alpha and L chains, that individual L chains probably can pair both covalently and non-covalently depending on the alpha chain.

Rapid structural characterisation of a murine monoclonal IgA alpha chain: heterogeneity in the oligosaccharide structures at a specific site in samples produced in different bioreactor systems.

A murine hybridoma cell line which secretes a monoclonal IgA antibody, directed against the LPS antigen of Vibrio cholerae, was grown in either a continuous stirred tank reactor, a fluidised bed reactor or a hollow fibre reactor. Different methods were used for the structural characterisation of the IgA alpha chains. A classical approach consisted of Edman sequencing and mass determination of peptides separated by reversed phase HPLC. Alternatively, peptides and glycopeptides from a tryptic digest of each alpha chain were identified directly by MALDI-TOF mass spectrometry. A detailed analysis of the oligosaccharide structures at an unique site on the alpha chain was made by labelling the oligosaccharides released by N-glycosidase F with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone. After separation by HPLC, mass measurements were made using matrix-assisted laser desorption time of flight mass spectrometry before and after digestion with specific exoglycosidases. The primary structure of the alpha chain of IgA was not affected by different cell culture conditions; in contrast, significant variations could be detected in the pattern of N-linked oligosaccharide structures, most prominently in the degree of sialylation. The efficiency of the analytical techniques in providing quality control of the identity, integrity and consistency of the glycoprotein is shown and discussed.

[Non-Mediterranean lymphoma producing heavy alpha chains]. / Linfoma no mediterráneo productor de cadenas pesadas alfa.

At the present, immunoproliferative small intestinal disease (IPSID) is considered a fairly homogeneous entity. Isolated heavy chain production is so closely related with IPSID that has been considered as a natural biologic marker for it. By contrast, we report here the case of a 63-year-old female that developed a multinodular small bowel lymphoma without clinical malabsorption symptoms. The main tumour mass was located in proximal jejunum and the neighbour intestinal mucosa did not show neoplastic cell infiltrates. Immunohistochemical methods demonstrated restrictive presence of IgA1 subclass in the neoplastic cells without presence of light chains. Cases as present are extremely infrequent and their potential relationship with IPSID has not been completely outlined, being useful in trying to understand the spectrum of gut lymphomas.

Transcription of a productively rearranged Ig VDJC alpha does not require the presence of HS4 in the IgH 3' regulatory region.

V gene assembly, class switch recombination, and somatic hypermutation are gene-modifying processes essential to the development of an effective Ab response. If inappropriately applied, however, these processes can mediate genetic changes that lead to disease (e.g., lymphoma). A series of control elements within the Ig H chain (Igh) locus has been implicated in regulating these processes as well as in regulating IgH gene transcription. These include the intronic enhancer (Emu) and several elements at the 3' end of the locus (hs1,2, hs3a, hs3b, and hs4) known collectively as the 3' regulatory region. Although it is clear that the Emu plays a unique role in V gene assembly, it has not been established whether there are unique functions for each element within the 3' regulatory region. In earlier studies in mice and in mouse cell lines, pairwise deletion of hs3b and hs4 had a dramatic effect on both class switch recombination and IgH gene transcription; deletion of an element almost identical with hs3b (hs3a), however, yielded no discernible phenotype. To test the resulting hypothesis that hs4 is uniquely required for these processes, we induced the deletion of hs4 within a bacterial artificial chromosome transgene designed to closely approximate the 3' end of the natural Igh locus. When introduced into an Ig-secreting cell line, an Igalpha transcription unit within the bacterial artificial chromosome was expressed efficiently and the subsequent deletion of hs4 only moderately affected Igalpha expression. Thus, hs4 does not play a uniquely essential role in the transcription of a productively rearranged Ig VDJCalpha transcription unit.

Immunoglobulin synthesis in the human myeloma cell line U-266; expression of two immunoglobulin heavy chain isotypes (epsilon and alpha) after long-term cultivation in vitro.

A human IgE-producing myeloma has been cultivated in vitro as a continuous cell line (U-266) since 1968. Analysis of immunoglobulin production during early passages of the cell line demonstrated a high synthesis rate of monoclonal IgE. Analysis of late passages, cultivated after 1980, revealed a 3-6-fold lower rate of IgE secretion. This decrease was accompanied by the appearance of small amounts of IgA in the culture medium together with IgE. RNA was extracted from a late passage of U-266 and analyzed by Northern blotting, using epsilon and alpha-specific oligonucleotides as hybridization probes. The results showed the presence of epsilon as well as alpha-specific mRNA. Moreover the results demonstrated that the latter mRNA was derived from the alpha 2 locus and that the epsilon and the alpha 2-specific mRNA contained the same V region sequences. Southern blot analysis of DNA from the late passage of the U-266 cell line failed to reveal a recombinatory switch from the epsilon locus to the alpha 2 locus. The expression of alpha 2 is thus likely to be caused by differential splicing rather than by an isotype switch at the DNA level.

Human lymphoid cells of the intestinal mucosa showing specificity for both immunoglobulin light and heavy chains.

The presence of single human lymphoid cells expressing on the one hand both kappa and lambda light chain and on the other hand either both mu and alpha or both mu and gamma heavy chains, was observed in the intestinal mucosa. The variability of the frequency of such cells, both from one subject to another, or in the same sample, is a characteristic of this population.

Synthesis and processing of the alpha heavy chains of secreted and membrane-bound IgA.

We have compared the synthesis and processing of immunoglobulin alpha chains in two murine cell lines, a B cell lymphoma that expresses membrane-bound IgA and a hybridoma that secretes IgA. Results of biosynthetic labeling experiments demonstrated that membrane-bound and secreted alpha chains have two distinct intracellular precursors, of different molecular weights and isoelectric points. RNAs from both of these cell lines direct the synthesis in vitro of two alpha polypeptides of Mr 59,000 and 62,000, the larger one being the precursor for membrane-bound alpha chain and the smaller one being the precursor for secreted alpha chain. These cell lines each contain three RNAs, 1.7, 2.1, and 3.1 kilobases in length, which hybridize with cDNA for the alpha constant region and which are present in different concentrations. Our results suggest that the smallest RNA encodes the secreted alpha chain and one or both of the larger RNAs encode(s) the membrane-bound alpha chain.

The mutual clonal origin of the lymphoplasmocytic and lymphoma cell in alpha-heavy chain disease.

Biosynthetic studies in alpha-heavy chain disease were performed on the gut tumour which was composed mainly of lymphoplasmocytic cells and on the mesenteric lymph node tumour composed mainly of immunoblasts. The gut tumour cells synthesised alpha-heavy chains and secreted them during 2-5 hr culture, whereas the lymph node tumour cells synthesized alpha-heavy chains which were shed into the culture medium only after 20 hr. These chains were shown to be present on the surface of the immunoblastic tumour cells by enzymatic radioiodination. Both the surface and the secreted alpha-heavy chain of the lymph node and gut tumour were found to be smaller than the alpha-heavy chain of myeloma proteins. These results suggest that the lymphoblasmocytic and the immunoblastic tumour cells originate from the same defective clone.

An unusual case of leukemia with high fetal hemoglobin: demonstration of abnormal hemoglobin synthesis localized in a red cell clone.

A high level of fetal hemoglobin was found in an 8-yr-old boy without any hematologic disorders except for a moderate anemia. The absence of hemoglobin abnormalities in the parents led us to suspect a latent malignant disease that, on follow-up, was confirmed to be myelomonocytic leukemia. Hemoglobin biosynthetic studies provided evidence of unbalanced synthesis of globin subunits by reticulocytes, while the production of non-alpha chains was equal to that of alpha chains in bone marrow cells. The expression of red cell antigen i was increased, while those of I, A, and A1 antigens were found to decrease progressively. Two populations of erythrocytes, A-positive and A-negative, were distinguished and could be separated by differential agglutination. Unbalanced globin chain synthesis, increased fetal hemoglobin, and antigenic changes of the membrane were shown to be restricted to the A-negative population. The biologic data were not entirely consistent with a genuine reversion to fetal erythropoiesis. The question remains of a polychromosomal lesion of either quiescent F cells or adult stem cells.

Regulation of transcription of the germ-line Ig alpha constant region gene by an ATF element and by novel transforming growth factor-beta 1-responsive elements.

Inasmuch as transcription of unrearranged, or germ-line, Ig CH genes appears to direct switch recombination, understanding the regulation of this transcription is essential for understanding the regulation of class switching. Transforming growth factor-beta 1 (TGF-beta 1) induces germ-line alpha transcripts and increases class switching to IgA in the I.29 mu B lymphoma and in Peyer's patch and splenic B cells. It has been previously demonstrated that induction of germ-line alpha transcripts by TGF-beta occurs at the transcriptional level in I.29 mu cells. We now demonstrate that the DNA segment located 5' to the initiation sites of germ-line alpha RNA drives expression of a luciferase reporter gene construct in transient transfection experiments. Full constitutive expression requires no more than 106 bp of the 5' flanking segment. By creating a series of deletion and substitution mutations, we have demonstrated that an ATF/CRE site residing within this region is very important for constitutive expression of the germ-line alpha promoter, but mutation of this motif does not diminish TGF-beta induction. Inducibility by TGF-beta requires additional sequences residing between -128 to -106 relative to the first RNA initiation site. Two copies of a tandemly repeated sequence 5' CA-CAG(G)CCAGAC 3' (termed Ig alpha TGF-beta-RE) are located in the region from -127 to -105. An oligonucleotide containing multimers of these repeats confers TGF-beta inducibility to a heterologous promoter. An additional copy of the TGF-beta-RE was identified at -41/-30 and its deletion reduces the TGF-beta response. Thus, we conclude that tandem repeats of a novel TGF-beta-RE are the positive regulatory element for the TGF-beta response. Our study provides further evidence that TGF-beta directs class switching to IgA through induction of transcription of the germ-line C alpha gene and demonstrates that TGF-beta can activate the promoter for the germ-line alpha gene.

Celluar immunoglobulins in human gamma- and alpha-heavy chain diseases.

Proliferating cells from twenty-four patients with alpha- or gamma-heavy chain disease (HCD) were studied by direct immunofluorescence and in several cases by biosynthesis experiments with 14C-amino acid incorporation. In twenty-two patients, the cells contained the HCD proteins only and no light chain synthesis could be detected. Conversely, apparently non-secreted monotypic light chains were found in one case of gamma-HCD and one case of alpha-HCD. The proportion of proliferating cells containing cytoplasmic heavy chains, their appearance and the presence or not of surface heavy chains showed great variation from patient to patient. In some cases, the proliferation predominantly affected either plasma cells or lymphocytes whereas in others the disease seemed to correspond to a proliferation of HCD protein-bearing lymphocytes with persistent maturation into plasma cells. Large cell lymphomas supervening on alpha-HCD belonged to the same proliferating clone as the clone secreting the HCD protein, as shown by surface markers and biosynthesis experiments which demonstrated synthesis but no secretion of HCD proteins. In one patient with gamma-HCD, the cells carried surface gamma and delta chains.

Synthesis but not secretion of J chain by variant mouse myeloma cells which lose alpha-chain-synthesizing ability.

We have devised a rapid method for obtaining large amounts of J chain from IgA in the ascitic fluid of mice bearing the MOPC 315 tumor. The J chain was released by reduction from the MOPC 315 IgA adsorbed onto a DNP-lysyl-Sepharose column, and was further purified by DEAE Sephadex chromatography. The mouse J chain was characterized as to its electrophoretic mobility, amino acid composition, apparent size, presence in different immunoglobulin classes, and reactivity with an antiserum containing anti-J chain activity. Variant cell lines have been selected from the IgA-producing mouse myeloma cell line MOPC 315. The variants did not synthesize detectable quantities of alpha heavy chains but continued to synthesize and secrete light chains. J chain was synthesized by both parent and variant cell lines but only secreted by the parent cells. It is postulated that J chain synthesis is not dependent on alpha heavy chain synthesis, but that secretion of J chain by MOPC 315 cells occurs only because of its attachment to the Ig1 molecule.

Hairy cell leukaemia occurring with an unrelated paraproteinaemia. A biochemical and immuno-electron microscopic study.

A detailed study is described of a case of hairy cell leukaemia, presenting with a serum paraprotein of an immunoglobulin (Ig) class different from that synthesised by the neoplastic cells. The case was unusual in its association with leukaemic arthropathy but ultrastructurally the hairy cells were typical. By immunofluorenscence and immuno-electron microscopy the neoplastic cells expressed IgA lambda both on the cell surface and intracellularly in the rough endoplasmic reticulum, perinuclear space and Golgi apparatus. No Ig was observed in the ribosomal-lamellae complexes. These cells also synthesised and secreted Ig of class A lambda in culture. However the serum paraprotein was of class IgA chi and could not be attributed to an abnormal population of plasma cells in the bone marrow. There was no other evidence for myeloma and the IgA chi paraproteinaemia appeared to be benign, apparently unrelated to the neoplastic proliferation of hairy cells.

Regulation of the antibody repertoire through control of HCDR3 diversity.

In man, as in mouse, diversification of the antibody repertoire appears to follow a strict developmental program whereby antigen specificities are serially acquired during ontogeny. When compared to the adult repertoire, the fetal antibody repertoire is highly enriched for polyreactive specificities of low affinity. Although the mechanisms governing the development of this fetal repertoire differ between human and mouse, the composition and structure of the fetal antibodies produced by both species are quite homologous. Specifically, both species use similar V gene segments and restrict the sequence and structure of the third complementarity determining region (HCDR3) of the antibody heavy chain. The precise role that this restriction of the HCDR3 might play in the development of immunocompetence in the human remains to be elucidated.

Immunobiology and pathogenesis of alpha chain disease.

alpha chain disease, the most frequent of the heavy chain diseases, is a proliferative disorder of B lymphoid cells involving primarily the small intestine and mesenteric nodes. The characteristic immunoglobulin, whose detection by immunochemical techniques may present some difficulties, consists of incomplete alpha chains devoid of light chains. The deleted portion of the alpha chain is located in the Fd segment and involves both the variable and first constant domains. In both of two proteins for which structural data are available, normal sequence resumes at the beginning of the hinge region. The absence of L chains is due to a failure of synthesis. alpha chain disease appears to proceed in two stages. The early stage is characterized by a possibly non-malignant diffuse and extensive plasma cell infiltration which may be reversible after administration of antibiotics. The later stage is characterized by overt neoplasia (immunoblastic lymphoma). The socio-geographic distribution of the digestive form of alpha chain disease shows a clear predilection for underpriviliged populations living in areas with a high degree of infestation by intestinal pathogens which play presumably a crucial role in the pathogenesis of the disease.