Harold D. Keiser

Prolactin alters the mechanisms of B cell tolerance induction

OBJECTIVE Autoimmune diseases predominantly affect women, suggesting that female sex hormones may play a role in the pathogenesis of such diseases. We have previously shown that persistent mild-to-moderate elevations in serum prolactin levels induce a break in self tolerance in mice with a BALB/c genetic background. The aim of this study was to evaluate the effects of hyperprolactinemia on the mechanisms of B cell tolerance induction. METHODS Effects of prolactin on splenic B cell subsets were studied in female BALB/c mice. B cell receptor (BCR)-mediated apoptosis and proliferation of transitional B cells were analyzed by flow cytometry. Expression of apoptotic genes was examined by microarrays and real-time polymerase chain reaction analysis. B cells coexpressing kappa/lambda light chains were assessed by flow cytometry and immunohistochemistry. Activation status of transitional type 3 (T3) B cells was evaluated by BCR-induced calcium influx studies. RESULTS BCR-mediated apoptosis of the T1 B cell subset, a major checkpoint for negative selection of autoreactive specificities, was decreased in prolactin-treated mice. Microarray studies indicated that this event may be mediated by the prolactin-induced up-regulation of the antiapoptotic gene interferon-gamma receptor type II and down-regulation of the proapoptotic gene Trp63. Prolactin treatment also altered the amount of receptor editing, as indicated by the increased number of transitional B cells coexpressing kappa/lambda light chains. Additionally, hyperprolactinemia modified the level of B cell anergy by increasing the degree of BCR-induced calcium influx in the T3 B cells. CONCLUSION Persistently elevated serum prolactin levels interfere with B cell tolerance induction by impairing BCR-mediated clonal deletion, deregulating receptor editing, and decreasing the threshold for activation of anergic B cells, thereby promoting autoreactivity.

Glycosaminoglycans Produced by Human Synovial Cell Cultures

Human synovial cells in culture are known to synthesize hyaluronic acid, but the production of sulfated glycosaminoglycans (GAG) has received less attention. Using 14C-glucosamine as a precursor, GAG content was studied in the medium, trypsin-solubilized pericellular layer, and cell residue fraction of cultured synovial cells derived from the synovial membranes of nonrheumatoid and rheumatoid joints. Over 90% of the total non-dialyzable counts appeared in the culture medium, for the most part in hyaluronic acid. The remaining nondialyzable counts were cell-associated, almost equally divided between the pericellular layer and cell residues. In these fractions, only part of the counts were in GAG susceptible to testicular hyaluronidase digestion, and GAG were significantly lower in the cell residue of the rheumatoid synovial cells compared to the nonrheumatoid cells. Analysis of the chondroitinase ABC and AC digestion products of these GAG indicated the presence of chondroitin-4 and -6 sulfates, and dermatan sulfate, but not heparan sulfate. Similar findings with respect to the identity of the GAG in nonrheumatoid and rheumatoid synovial cell culture media were obtained with 35SO4 as a precursor.

Immunochemical Studies of Fragments of Bovine Nasal Cartilage Proteoglycan Subunit

Cartilage proteoglycan subunit, which consists of a core protein with chondroitin sulfate and keratan sulfate side chains, contains an antigen common to many species. In order to better characterize this antigen, fragments of bovine nasal cartilage proteoglycan subunit were produced by proteolytic digestion of the core protein and these were separated into chondroit in sulfate and keratan sulfate-containing pept ides by DEAE-cellulose chromatography. Immunochemical studies of these fragments indicate that proteoglycan subunit is not uniform in its structure and that its antigenicity is due to determinants in the core protein, particularly in those segments of the core protein near the attachment of keratan sulfate side chains.

Relapsing arterial aneurysms in juvenile Behçet’s disease

Vascular involvement in Behçet’s disease has a wide range of clinical manifestations, the most common being thrombophlebitis and arterial aneurysms. Every major vessel may be involved, but aneurysms of the carotid artery are relatively rare in adults and even rarer in children. We describe here a case of a 13-year-old boy with Behçet’s disease with a recurrent carotid artery aneurysm treated with arterial ligation and immunosuppressive therapy who later developed an abdominal aortic aneurysm in the absence of other indications of disease activity. This case underscores the need for continuous monitoring of patients with vascular manifestations of Behçet’s disease.

Monoclonal Antibodies Reacting with Endo-β-Galactosidase-Resistant Epitopes on Keratan Sulfate-Bearing Fragments of Bovine Nasal Cartilage Proteoglycan

Six monoclonal antibody-producing cell lines were derived from mice immunized with keratan sulfate (KS)-bearing tryptic fragments of the core protein of bovine nasal cartilage proteoglycan monomer digested with KS-specific endo-beta-galactosidase. The monoclonal antibodies were characterized by solid-phase ELISA competition studies and SDS-PAGE immunoblotting. Two of them resemble previously described monoclonal antibodies that are directed to epitopes containing both KS and core protein. In contrast, the remaining four monoclonal antibodies are unprecedented in being directed to epitopes whose reactivity is unaffected or enhanced by endo-beta-galactosidase degradation of KS. SDS-PAGE immunoblots revealed two large KS-bearing tryptic fragments of cartilage proteoglycan and a heterogeneous population of smaller fragments not evident by non-immunological techniques. Some of the antibodies used react with all KS-bearing fragments, others react only with the two largest fragments.

A comparison of the proteoglycans produced by rabbit articular chondrocytes in monolyayer and spinner culture and those of bovine nasal cartilage

The structural and immunological properties of the glycosaminoglycans and the core proteins of bovine nasal cartilage proteoglycan and the proteoglycans produced by rabbit articular chondrocytes in spinner and monolayer culture were compared. Culture medium with 35SO4- or 3H-serine-labeled proteoglycan was mixed with bovine nasal cartilage 4M guanidine-HCl extract and digested with trypsin. The proteoglycan fragments were then isolated by DEAE-cellulose chromatography and fractionated by dissociative CsCl density gradient centrifugation. Approximately 90% of the 35SO4 incorporated into proteoglycan by the cultured chondrocytes was in chondroitin sulfates and about 5% in keratan sulfate. Although there was considerable overlap in the Sepharose 4B elution of the tryptic proteoglycan fragments of highest buoyant density, some monolayer-produced proteoglycan fragments eluted earlier and some spinner-produced proteoglycan fragments eluted later than the proteoglycan fragments from bovine nasal cartilage. These differences in apparent fragment size could relate to differences in glycosaminoglycan chain length, since the glycosaminoglycans released by treatment with alkali from monolayer-produced proteoglycan in part eluted from Sepharose 4B earlier and those from spinner-produced proteoglycan in part eluted later than the chondroitin sulfate chains released from bovine cartilage proteoglycan. After digestion with chondroitinase ABC, 3H-serine-labeled high density tryptic proteoglycan fragments from monolayer and spinner culture yielded Sepharose 6B elution profiles which were similar to each other but did not coincide with the peaks of carbazole reactivity found with similarly treated fragments of bovine nasal cartilage proteoglycan. Cross-reactivity was demonstrated by radioimmunoautography between bovine cartilage and rabbit chondrocyte proteoglycan fragments restricted to gradient fractions of low buoyant density, but immunological cross-reactivity was not found for the antigens associated with the keratan sulfate-rich and chondroitin sulfate-bearing tryptic fragments of bovine nasal cartilage proteoglycan. These studies indicate that the proteoglycan core proteins produced by rabbit articular chondrocytes in monolayer and spinner culture are, in part, different from the core protein of bovine nasal cartilage proteoglycan and that the three proteoglycans differ in the length of some of their chondroitin sulfate chains.

Prolactinoma and systemic lupus erythematosus : do serum prolactin levels matter?

The lactogenic hormone prolactin is produced in part by cells of the immune system and serves as an upregulator of immune function. Hyperprolactinemia is common in patients with systemic lupus erythematosus (SLE), raising the possibility that the hormone contributes to the excessive immune response in the disease. The highest levels of circulating prolactin occur in association with prolactin-secreting tumors, but prolactinomas have only rarely been encountered in patients with SLE. We present here three patients with SLE and prolactinomas. As with the previously reported six patients, there was no consistency in the presence of findings related to prolactin excess or in the coincidence of hyperprolactinemia with flares of SLE disease activity. We speculate that this may be due to genetic differences in the response to prolactin and/or to the presence of variant prolactin isoforms detected in the clinical immunoassay that have reduced or absent biologic activity.

A solid-phase immunoassay for the binding of cartilage proteoglycan to hyaluronic acid.

A solid-phase assay for detecting the binding of cartilage proteoglycan (PG) to hyaluronic acid (HA) is described. In the assay, HA is immobilized on protamine-treated microtiter wells, the wells are incubated with PG monomer and antibody to PG monomer, and then an ELISA system is used to detect binding of the PG to HA. The specificity of the assay is indicated by the failure to detect PG binding to chondroitin sulfate or albumin-coated microtiter wells, the absence of binding with tryptic fragments of PG monomer other than the HA-binding segment, the loss of binding after reduction and alkylation of PG monomer, and the inhibition of binding by preincubation of PG monomer with small amounts of HA. In contrast to the HA-PG interaction in solution, hyaluronidase digestion of HA does not affect its ability to inhibit the reaction of PG monomer with immobilized HA. The microtiter well-based assay appears to be a rapid, simple, and potentially versatile method for studying interactions with HA.

Inhibition of Adenovirus DNA Synthesis In Vitro by Sera from Patients with Systemic Lupus Erythematosus

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60 degrees C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases alpha, beta, gamma and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.

The Immunology of Proteoglycans

This discussion will touch on some of the central features of proteoglycan (PG) immunology and suggest that despite the recent introduction of very sophisticated immunochemical techniques, this remains a problematic field. As I shall indicate, there are difficulties in understanding the immunological properties of PGs which are due to the nature of PG, to a basic uncertainty of the details of PG structure and organization and, finally, to our inability to isolate truly homogeneous PGs or PG fractions for use in immunological studies. The earliest work in PG immunology showed that although the glycosaminoglycan (GAG) portion of the PG comprises the bulk of its composition and determines its physicochemical properties, isolated chondroitin sulfate (CS) or keratan sulfate (KS) chains are immunologically silent. Immunization of experimental animals with the isolated GAGS does not lead to the production of antibodies detectable by immune precipitation, hemagglutination inhibition or cutaneous reactivity. Furthermore, GAG chains isolated by (3-elimination or papain digestion of PG do not influence the binding of multivalent anti-PG sera with PG radio-labeled either in the core protein or in the CS Thus, it appears that the immunogenicity of PG is due to its protein constituents. This has both good and bad consequences. The chemical and enzymatic methods for the analysis of the GAG chains are well established and our knowledge of their structure and synthesis is well developed, whereas our knowledge of the detailed structure of the PG core protein is limited. The specificity of anti-PG antibodies for PG protein offers a potentially valuable direct probe for the study of PG proteins in face of the relatively small amount present in PG. On the other hand, in trying to understand and interpret the immunological findings, we are sorely handicapped by our incomplete knowledge of what the PG proteins are and how they fit together. A second early discovery of PG immunology was that the PG molecule must be modified in order to allow it to be studied. Treatment of cartilage sections or of the extracted chondromucoprotein or proteinpolysaccharide, as PG was then known, with testicular hyaluronidase was found necessary in order to demonstrate full immunological reactivity, even though the antisera used were produced by immunization with PG fractions which had not been digested with hyal~ronidase.~* ~*~ The explanations offered for this were that PG molecules are just too big to diffuse through agarose, so they can’t be identified by Ouchterlony immunodiffusion, or that the CS chains are so big and bulky that the antibody molecules can not get near the antigen.3 I believe that the observation is correct, but I am not sure about the explanations. PG molecules are quite capable of diffusing through agarose and many of us make our livings by characterizing this process. In addition, if PG molecules are digested with trypsin, which does not affect the CS chains that are