Light chain usage in anti-double-stranded DNA B cell subsets: role in cell fate determination.

Two major mechanisms for the regulation of autoreactive B cells that arise in the bone marrow are functional silencing (anergy) and deletion. Studies to date suggest that low avidity interactions between B cells and autoantigen lead to B cell silencing, whereas high avidity interactions lead to deletion. Anti-double stranded (ds) DNA antibodies represent a pathogenic autospecificity in Systemic Lupus Erythematosus (SLE). An understanding of their regulation is critical to an understanding of SLE. We now demonstrate in a transgenic model in which mice express the heavy chain of a potentially pathogenic anti-DNA antibody that antibody affinity for dsDNA does not alone determine the fate of anti-dsDNA B cells. B cells making antibodies with similar affinities for dsDNA are regulated differently, depending on light chain usage. A major implication of this observation is that dsDNA may not be the self antigen responsible for cell fate determinations of anti-dsDNA B cells. Light chain usage may determine antigenic cross-reactivity, and cross-reactive antigens may regulate B cells that also bind dsDNA.

Bromocriptine restores tolerance in estrogen-treated mice.

Estrogen can modulate autoimmunity in certain models of systemic lupus erythematosus. Recently, we have shown that it can mediate survival and activation of anti-DNA B cells in a mouse transgenic for the heavy chain of a pathogenic anti-DNA antibody. To identify whether estrogen effects reflect increased prolactin secretion, we characterized B-cell autoreactivity in transgenic mice given both bromocriptine (an inhibitor of prolactin secretion) and estradiol. Treatment of mice with estradiol plus bromocriptine led to reduced titers of anti-DNA antibodies and diminished IgG deposition in kidneys compared with treatment with estradiol alone. However, mice treated with estradiol plus bromocriptine showed an expansion of transgene-expressing B cells and enhanced Bcl-2 expression, similar to those of estradiol-treated mice. We identified anergic high-affinity anti-DNA B cells in mice treated with estradiol plus bromocriptine, and we showed by molecular analysis of anti-DNA hybridomas that their B cells derive from a naive repertoire. Thus, the estradiol-induced breakdown in B-cell tolerance can be abrogated by bromocriptine, which induces anergy in the high-affinity DNA-reactive B cells. These studies demonstrate that some of the effects of estrogen on naive autoreactive B cells require the presence of prolactin and, thus, suggest potential therapeutic interventions in lupus.

DNA sequence analysis and comparison of the variable heavy and light chain regions of two IgM, monoclonal, anti-myelin associated glycoprotein antibodies.

The complete variable heavy and light chain gene sequences of two monoclonal, IgM, anti-myelin associated glycoprotein (MAG) antibodies associated with peripheral neuropathy, are presented. Comparative analysis of the two VH regions has revealed that they are 88% homologous to one another and are both members of the VH3 gene family. They are also highly homologous to a gene which is frequently utilized in the fetal B-cell repertoire. The V kappa light chain gene of one of the antibodies is 99% homologous to a V kappa II gene and the V lambda light chain gene of the other antibody is only 72% homologous to other known V lambda genes. Further analysis of V genes utilized by anti-MAG antibodies should reveal the structural basis for their binding activity.

The B-cells that express anti-MAG antibodies in neuropathy and non-malignant IgM monoclonal gammopathy belong to the CD5 subpopulation.

B-cells that express monoclonal IgM anti-MAG antibodies (M-protein) in a patient with neuropathy and non-malignant IgM monoclonal gammopathy were studied by flow cytometry to determine if they bear the CD5 cell surface antigen. The CD5+ antigen is expressed on a subpopulation of B-cells which has been implicated in the secretion of autoantibodies and development of B-cell chronic lymphocytic leukemia (B-CLL). The monoclonal anti-MAG antibodies secreting B-cells were identified by a mouse monoclonal antibody to M-protein idiotype. Cytofluorometric analysis revealed that approximately 62% of the idiotype-positive B-lymphocytes expressed the CD5 antigen. The result of this study indicate that monoclonal anti-MAG antibodies may be secreted by CD5+ B-cells, and that B-cells in some cases of non-malignant IgM monoclonal gammopathy may be phenotypically similar to B-CLL cells.

Molecular cloning of a human immunoglobulin heavy chain variable (VH) region with anti-myelin-associated glycoprotein activity.

A cDNA clone that encodes the heavy chain variable region (VH) of an IgM M-protein with anti-myelin-associated glycoprotein (MAG) activity secreted by chronic lymphocytic leukemia cells (B-C11) from a patient with peripheral neuropathy was cloned and sequenced. The JH region was identical to the germline JH4 sequence except for deletion of a thymidine residue at the site of D-JH recombination, and the D region showed greatest homology to DM2. Sequence analysis of the VH region revealed greatest homology to VH26, a member of the VH3 gene family, but homology was only 83.7% over 326 bases, suggesting that it was derived from as yet an unidentified member of the VH3 gene family.

Neutrophilic hypersegmentation as an indicator of incipient folic acid deficiency.

The authors have identified a group of subjects with neutrophilic hypersegmentation who are normal or near-normal with respect to other hematologic indices (hemoglobin, mean corpuscular volume). In a high proportion of these subjects, serum folate levels are abnormally low. In this group and a non-hypersegmented-neutrophil control group there was a significant negative correlation between average numbers of neutrophilic lobes and serum folate levels. In the subjects with hypersegmented neutrophils the predominant alteration is a shift from three-lobed to five-lobed neutrophils. It is believed that neutrophilic hypersegmentation can be a valuable adjunct in documenting and/or uncovering incipient folate deficiency.

Myelination by transplanted fetal and neonatal oligodendrocytes in a dysmyelinating mutant.

Demyelination is a major feature of CNS injury and disease, including multiple sclerosis. To examine the potential for myelination by transplanted oligodendrocytes, initially described by Gumpel et al., we have transplanted neonatal cortex of mice with normal myelin into a dysmyelinating mutant, the shiverer mouse. We have found that oligodendrocyte precursors mature and synthesize myelin following transplantation. Immunostaining with antibodies to myelin basic protein (MBP), neurofilament protein and glial fibrillary acidic protein, demonstrates myelination both within the graft and extending out into the host, axonal sprouting from the graft which parallels the MBP-reactivity, and minimal astrocytic proliferation in response to the transplant.

Creatine kinase (CK) isoenzyme activities in cardiac surgery.

Creatine kinase (CK) isoenzymes were determined on 15 patients undergoing open heart surgery with bypass. The study was performed to examine the relationship between the hypothermia under which the surgery is conducted and the appearance of CK isoenzymes in the serum both during and after surgery. All patients showed dramatic rises in total CK activity commencing during surgery. Myocardial CK (CK-MB or CK-2) was seen in fourteen patients and brain CK (CK-BB or CK-1) was seen in ten patients. Peak activities of CK-BB did not coincide with peak activities of CK-MB. The serum elevations of CK-BB in these patients appear to arise from a mechanism different from that responsible for the elevations of CK-MB, and it is assumed that the former is due to intermittent disruptions in the perfusion and/or oxygenation of tissues rich in CK-BB. CK-MB elevations appear to be due directly to the surgical intervention. Hypothermia alone does not in itself appear to be solely responsible for elevations of CK-BB although it can not be completely excluded from playing some role in its production.

New intermediate hosts in the life cycle of Zygocotyle lunata in South America.

Biomphalaria peregrina was found to be naturally infected with cercariae of Zygocotyle lunata in a pond of the Zoological Garden of Buenos Aires. Mice and chicks were fed metacercariae and gravid adults recovered. Eggs recovered from mice feces were used for experimental infections. Laboratory-reared B. peregrina and 4 other Biomphaluria spp. were successfully infected with Z. lunata miracidia. Biomphaleria glabrata was refractory. Species of Helisoma, the only intermediate hosts of Z. lunata so far reported, have never been found in Argentina. Species of Biomphalaria may be intermediate hosts of Z lunata in the southern region of the Parana River Basin.

A form of cytochrome b5 that contains an additional hydrophobic sequence of 40 amino acid residues.

A species of cytochrome b(5) with a monomer molecular weight of 16,700 has been isolated from rabbit-liver microsomes by a procedure that uses detergents and avoids the use of any proteolytic or lipolytic enzymes. This detergent-extracted cytochrome b(5) is larger than the trypsin- or lipase-extracted enzyme, and appears to contain an extremely hydrophobic appendage of 40 amino acids, probably at the N-terminus. The hydrophobic character of the extra amino acid sequence leads to aggregation in the absence of detergents, and may be of considerable importance in the binding of the enzyme to microsomes. It is suggested that the hydrophilic portion of the cytochrome molecule, which bears the heme and is enzymatically functional, is oriented toward the surface of the membrane where it readily reacts with nonmicrosomal proteins, while the hydrophobic "tail" anchors the heme protein tightly to the membrane.

Secretions of anti-myelin-associated glycoprotein antibodies by B cells from patients with neuropathy and nonmalignant monoclonal gammopathy.

Four patients with peripheral neuropathy and nonmalignant monoclonal gammopathy with anti-myelin-associated glycoprotein (MAG) antibodies were studied to determine whether secretion of anti-MAG IgM antibodies by B cells was autonomous, or whether the monoclonal B cells were responsive to T cells. Secretion of anti-MAG IgM by isolated B cells was stimulated by the addition of increasing numbers of pokeweed mitogen (PWM)-activated autologous OKT4+ helper T cells in all four patients. Secretion of anti-MAG IgM by peripheral blood lymphocytes was dependent on the ratio of OKT4+ T helper cells to OKT8+ T suppressor/cytotoxic cells. In three patients with an OKT4+ to OKT8+ T-cell ratio of 2:1, PWM activation stimulated secretion of anti-MAG IgM; in one patient with an OKT4+ to OKT8+ ratio of 1:2, activation by PWM suppressed anti-MAG IgM secretion. These studies suggest that the monoclonal B cells that secrete anti-MAG IgM are responsive to regulatory T cells.

Lack of allelic exclusion permits autoreactive B cells to escape deletion.

The R4A-gamma 2b transgenic mouse harbors the gene for the gamma 2b heavy chain of an anti-dsDNA Ab. Approximately 80% of B cells expressing the transgene display allelic exclusion. Although the transgenic mice have little to no detectable serum anti-DNA activity, splenic B cells can be stimulated in vitro with LPS to secrete anti-DNA Ab. Hybridomas derived from LPS-stimulated splenic B cells were analyzed for expression of the transgene and for DNA binding. All nine transgene-encoded anti-DNA-producing lines were found to express an endogenous IgM heavy chain. Of 19 randomly selected lines producing a transgene-encoded non-DNA binding Ab, none expressed a second heavy chain. The tight correlation between lack of allelic exclusion and anti-dsDNA specificity provides strong support for the hypothesis that a major function of allelic exclusion is to prevent the maintenance of a pool of potentially activatable autoreactive cells.

Characterization of anti-DNA B cells that escape negative selection.

One of the challenges in the study of autoimmunity is to understand which autoreactive cells are subject to regulation and what mechanisms of regulation are operative. In mice transgenic for the R4A-gamma2b heavy chain of an anti-double stranded (ds) DNA antibody, the gamma2b heavy chain can pair with the full spectrum of endogenous light chains to produce a multitude of antibodies, including anti-dsDNA antibodies of different affinities and fine specificities. We have previously demonstrated the existence of two populations of anti-DNA B cells in non-autoimmune hosts: a high-affinity population which is rendered anergic in vivo, and a second high-affinity population which is deleted. We have now identified a third population of dsDNA-binding B cells. These cells produce germ-line-encoded antibodies with an apparent affinity for dsDNA that is 1 to 4 logs lower than the apparent affinities of antibodies made by anergic or deleted B cells, and represent a non-tolerized population which escapes regulation. Based on its characterization, we can define a molecular threshold for tolerance induction, and can speculate on the fate of these B cells when they are recruited to an immune response and undergo somatic mutation to become high-affinity anti-DNA B cells.

Induction of tolerance to an IgG autoantibody.

Nonautoimmune mice transgenic for the heavy chain of an IgG2b anti-double-stranded-DNA antibody express the transgene in lymphoid organs and display partial allelic exclusion of this gamma 2b transgene. The spleens of these mice are characterized by marked B-cell depletion. Although there are B cells in these mice that express the transgene and recognize double-stranded DNA, they are anergic in vivo. Recovery from the state of anergy occurs in vitro after lipopolysaccharide stimulation. Thus this transgenic model demonstrates the induction of self tolerance to an IgG autoantibody.

Expression of recombinant human anti-MAG antibodies in non-lymphoid mammalian cells.

The variable heavy and light chain genes of a monoclonal, IgM, anti-MAG antibody from a patient with neuropathy were inserted into expression vectors containing the gamma and kappa constant regions respectively and co-transfected into monkey kidney CV1P cells. The expressed antibody had the same antigenic specificity but significantly lower avidity than the native IgM, anti-MAG, antibody as detected by ELISA. When the variable heavy chain gene of the anti-MAG antibody was co-transfected with the variable light chain gene from another monoclonal, IgM, anti-MAG antibody, a fully assembled antibody was expressed as determined by a trapping ELISA, but it did not bind to (MAG) or to sulfated glucuronic acid paragloboside, indicating that both heavy and light chains contribute to the binding activity.

Generation of human B-cell hybridomas secreting monoclonal anti-myelin-associated glycoprotein antibodies from a patient with neuropathy.

Human hybridomas that secrete monoclonal IgM anti-myelin-associated glycoprotein antibodies were generated by fusion with cells from a patient with peripheral neuropathy and IgM monoclonal gammopathy. Karyotypic analysis of the hybridoma cells revealed no chromosomal abnormalities. The cells were positive for cell-surface idiotype HLA-DR and the plasma cell antigen PCA-1, and negative for the B-cell determinant B4 and for Leu-1, which has been postulated to distinguish a subpopulation of B cells that secrete IgM with autoantibody activity.

Peptide inhibition of glomerular deposition of an anti-DNA antibody.

Antibodies to double-stranded DNA are pathognomonic of systemic lupus erythematosus and deposit in the kidneys of lupus patients to cause glomerulonephritis. Recent data suggest that a significant proportion of anti-DNA antibodies may cross-react with renal antigens and be sequestered in the kidney by virtue of this cross-reactivity. If this is true, antigenic competition for pathogenic antibodies might prevent their deposition in kidneys and the ensuing tissue damage. To generate surrogate antigens that could be used for this purpose, we have used peptide display phage libraries to identify peptides that react with R4A, a pathogenic mouse monoclonal anti-DNA antibody that deposits in glomeruli. We have demonstrated that the peptides bind in or near the double-stranded DNA binding site. Furthermore, the peptides are bound preferentially by the R4A antibody as compared with two closely related antibodies derived from it, one of which deposits in renal tubules and one of which displays no renal pathogenicity. Administration of one of these peptides in a soluble form protects mice from renal deposition of the R4A anti-DNA antibody in vivo. This represents a new therapeutic approach in systemic lupus erythematosus that focuses on protecting target organs from antibody mediated injury.