Regional variation in the lamina propria T cell receptor V beta repertoire in normal human colon

The Ewing's sarcoma cell line RD-ES, which carries the EWS/FLI-1 fusion gene, responded to the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin with growth arrest. Replenishment of mevalonate (MVA) to the arrested cells restored cell growth. However, if tunicamycin (TM), which is an inhibitor of N-linked glycosylation, was present together with MVA the cells remained arrested, indicating that N-linked glycosylation is of importance for growth of Ewing's sarcoma cells. Inhibition of the biosynthesis of EWS/FLI-1 fusion protein by treatment with antisense oligonucleotides also led to growth arrest, suggesting that this protein is of importance for cell growth. We investigated whether MVA synthesis and N-linked glycosylation could be involved in regulation of the expression of the EWS/FLI-1 fusion protein, which in fact contains four potential sites for N-linked glycosylation. We found that inhibition of both HMG-CoA reductase and N-linked glycosylation drastically decreased the expression of the fusion protein, which mainly appears in the cell nuclei. There was no significant difference in the inhibitory effect on the fusion protein between the cytoplasm and the cell nuclei, indicating that the transport of the fusion protein to the cell nucleus is not affected. The fusion protein did not exhibit any gel electrophoretic mobility shift after treatment of the cells with lovastatin or TM, and it did not incorporate [3H]glucosamine. Therefore we can conclude that the fusion protein is not a glycoprotein. The decreased expression of the fusion protein following lovastatin or TM treatment was found to be due to a lowered stability of de novo-synthesized fusion protein. The down-regulation of the fusion protein was correlated to growth arrest. Furthermore, the kinetics between the expression of EWS/FLI-1 fusion protein and the initiation of DNA synthesis in MVA-stimulated cells were similar. Taken together, our data suggest that the regulatory role of N-linked glycosylation in the expression of the EWS/FLI-1 fusion protein is important for growth of Ewing's sarcoma cells. Possible mechanisms underlying TM-induced decrease in EWS/FLI-1 expression may involve the breaking of growth factor receptor pathways.     

Targeted disruption of the V(H) 81X gene: influence on the B cell repertoire

We have generated a mutant mouse in which the most D-proximal V(H) gene (V(H)81X) has been disrupted by introducing a neomycin-resistance gene into the V(H)81X exon by means of gene targeting in embryonic stem cells. The mutant mice generated are unable to express the V(H)81X gene but appear to display a normal pattern of B cell differentiation as well as normal numbers of bone marrow and peripheral B cells from fetal life all through ontogeny. They mount normal immune responses to several different antigens tested. In contrast, the distribution of V(H) gene rearrangements in the V(H)7183 family is altered in homozygous mutant mice. Thus, the antibody repertoire of the targeted mice is modified, at least as far as the expression of V(H)7183 genes is concerned.     

Generation of the germline peripheral B cell repertoire: VH81X-lambda B cells are unable to complete all developmental programs

The generation of VH81X heavy chain lambda-light chain-expressing B cells (VH81X-lambda+ B cells) was studied in VH81X heavy chain transgenic mice as well as in VH81X JH (-/-) and VH81X JH (-/-) Ck (-/-) mice, in which competition resulting from expression of heavy and light chains from the endogenous heavy and kappa light chain loci was prevented. We show that although lambda light chain gene rearrangements occur normally and give rise to light chains that associate with the transgenic heavy chain to form surface and soluble IgM molecules, further B cell development is almost totally blocked. The few VH81X-lambda+ B cells that are generated progress into a mature compartment (expressing surface CD21, CD22, CD23, and low CD24 and having a relatively long life span) but they also have reduced levels of surface Ig receptor and express higher amounts of Fas Ag than VH81X-kappa+ B cells. These VH81X-lambda+ B cells reach the peripheral lymphoid organs and accumulate in the periarteriolar lymphoid sheath but are unable to generate primary B cell follicles. In other heavy chain transgenic mice (MD2, M167, and M54), lambda+ B cells are generated. However, they seem to be preferentially selected in the peripheral repertoire of some transgenic heavy chain mice (M54) but not in others (MD2, M167). These studies show that a crucial selection step is necessary for B cell survival and maintenance in which B cells, similar to T cells, receive signals depending on their clonal receptors.     

Nonsteroidal anti-inflammatory agents: potential modifiers of periodontal disease progression

The most recent studies of NSAIDs as potential modifiers of periodontal disease progression are reviewed. These studies indicate that NSAIDs have the ability to alter or control the progression of periodontal disease. Reductions in gingival inflammation and in bone loss have been observed following the administration of various NSAIDs. In some cases, bone gain has been achieved. More research is required to determine the possible long-term side effects associated with the chronic usage of NSAIDs, as well as to achieve consensus on the most effective drug for the control of periodontal disease. Until this occurs, and this application of NSAIDs receives government approval, the use of these drugs to control periodontal disease is not recommended.     

The repertoire of rheumatoid factor-producing B cells in normal subjects and patients with rheumatoid arthritis

OBJECTIVE: To compare the B cell repertoire of normal individuals and patients with rheumatoid arthritis (RA) and, specifically, to identify precursor B cells with the potential to secrete rheumatoid factor (RF) and to understand the T helper cell requirements for the production of this autoantibody. METHODS: Frequencies of precursors of IgM-, IgG-, and RF-producing B cells were measured in a limiting-dilution system. Two distinct sources of T cell help were compared. T cell help was provided by anti-CD3-activated CD4+ human T cell clones, or T cell-B cell interaction was facilitated by the bacterial super-antigen staphylococcal enterotoxin D (SED). RESULTS: A subset of 2-14% of peripheral blood B cells secreted IgM and IgG in SED-driven cultures. The SED-responsive B cell subpopulation was present at 10 times higher frequency in normal donors compared with RA patients. However, the repertoires were very similar, particularly for RF+ precursors, which represented approximately one-third of all SED-responsive B cells. In normal individuals, most of these RF+ precursor B cells did not respond to anti-CD3-activated T helper cells, with only a very small fraction of B cells activated by anti-CD3-driven helper cells maturing into RF-secreting B cells (from 1 of 182 to 1 of 889 IgM-producing B cells). This subset was expanded approximately 50-fold in RA patients. CONCLUSION: Normal subjects and RA patients share a pool of B cells which secrete RF when activated in the presence of SED and T helper cells. These B cells are frequent and obviously anergic in normal individuals. The B cell subset with the potential to produce RF when help is provided in noncognate T-B interaction (anti-CD3-driven T cells) is considerably expanded in RA patients, probably reflecting an increased responsiveness of such B cells to helper signals.     

Phosphodiesterase profile of human B lymphocytes from normal and atopic donors and the effects of PDE inhibition on B cell proliferation

1. CD19+ B lymphocytes were purified from the peripheral blood of normal and atopic subjects to analyse and compare the phosphodiesterase (PDE) activity profile, PDE mRNA expression and the importance of PDE activity for the regulation of B cell function. 2. The majority of cyclic AMP hydrolyzing activity of human B cells was cytosolic PDE4, followed by cytosolic PDE7-like activity; marginal PDE3 activity was found only in the particulate B cell fraction. PDE1, PDE2 and PDE5 activities were not detected. 3. By cDNA-PCR analysis mRNA of the PDE4 subtypes A, B (splice variant PDE4B2) and D were detected. In addition, a weak signal for PDE3A was found. 4. No differences in PDE activities or mRNA expression of PDE subtypes were found in B cells from either normal or atopic subjects. 5. Stimulation of B lymphocytes with the polyclonal stimulus lipopolysaccharide (LPS) induced a proliferative response in a time- and concentration-dependent manner, which was increased in the presence of interleukin-4 (IL-4). PDE4 inhibitors (rolipram, piclamilast) led to an increase in the cellular cyclic AMP concentration and to an augmentation of proliferation, whereas a PDE3 inhibitor (motapizone) was ineffective, which is in accordance with the PDE profile found. The proliferation enhancing effect of the PDE4 inhibitors was partly mimicked by the cyclic AMP analogues dibutyryl (db) cyclic AMP and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, Sp-isomer (dcl-cBIMPS), respectively. However, at concentrations exceeding 100 microM db-cyclic AMP suppressed B lymphocyte proliferation, probably as a result of cytotoxicity. Prostaglandin E2 (PGE2, 1 microM) and forskolin (10 microM) did not affect B cell proliferation, even when given in combination with rolipram. 6. Inhibition of protein kinase A (PKA) by differentially acting selective inhibitors (KT 5720, Rp-8-Br-cyclic AMPS) decreased the proliferative response of control cells and reversed the proliferation enhancing effects of rolipram. 7. Importantly, PDE4 activity in LPS/IL-4-activated B lymphocytes decreased by about 50% compared to unstimulated control values. 8. We conclude that an increase in cyclic AMP, mediated by down-regulation of PDE4 activity, is involved in the stimulation of B cell proliferation in response to LPS/IL-4. B cell proliferation in response to a mitogenic stimulus can be further enhanced by pharmacological elevation of cyclic AMP.     

Regulation of the mature human T cell receptor gamma repertoire by biased V-J gene rearrangement

Vacuolar apical compartment (VAC) is a transient organelle originally observed in Madin-Darby canine kidney (MDCK) epithelial cells impaired from forming cell-cell contacts. VACs are large vacuoles which contain microvilli and apical plasma membrane markers (among others, a 184-kDa plasma membrane protein, AP2), but exclude basolateral membrane markers. Upon reestablishment of cell-cell contacts, VACs are rapidly (within 1 h) exocytosed toward intercellular spaces, after which the apical plasma membrane drifts toward its final destination (Vega-Salas, Salas, and Rodriguez-Boulan. 1988. J. Cell Biol. 107, 1717-1728). In this work, we studied the role of cAMP as a mediator for the exocytosis of VACs. We shifted confluent cells from low to normal calcium medium (thus reestablishing cell-cell contacts and causing VAC exocytosis), a shift which resulted in a significant rise of cellular levels of both total intracellular and protein-bound cAMP. The 8-Br analog of cAMP (8-Br-cAMP) (5-50 microM) caused externalization of the intracellular compartment of AP2 as measured by radioimmunoassay. A similar effect was observed with 3-isobutyl-1-methylxanthine. 8-Br-cAMP also caused the appearance of AP2-positive VAC images in nonpermeabilized cells, namely, VACs that become accessible to extracellular antibodies upon fusion with the plasma membrane. Lanthanum, which abolishes the peak of intracellular free calcium during a calcium switch, failed to block the exocytosis. On the other hand, 12-O-tetradecanoylphorbol-13-acetate induced only a modest exocytic response. Finally, 8-Br-cAMP induced VAC exocytosis in sparse MDCK cells grown in normal calcium medium. These data indicate that cAMP is a mediator between the extracellular signal provided by cell-cell contacts and VAC exocytosis.     

T helper cell regulation of normal and neoplastic B cell growth

Following challenge with a thymus-dependent antigen T helper cells regulate B cell growth and differentiation in several ways. Initially, the T cells physically associate with antigen presenting B cells. While in conjugate, the two cells communicate with each other through the actions of cell surface receptors whose ligands are integral membrane proteins expressed on the surface of the apposed cell. The ensuing biochemical pathways regulate the expression of genes required for B cell cycle progression. As a consequence of this interaction, the T cells are induced to synthesize and secrete soluble mediators that also affect B cell proliferation, as well as determining the ultimate fate of the activated B cell. We also suggest a role for normal, Th cells during the development, and continued expansion of certain types of B cell lymphomas.     

Altering the antibody repertoire via transgene homologous recombination: evidence for global and clone-autonomous regulation of antigen-driven B cell differentiation

Antibody VH transgenes containing small amounts of natural 5' and 3' flanking DNA undergo nonreciprocal homologous recombination with the endogenous Igh locus in B cells. The resulting "hybrid" heavy chain loci are generated at a low frequency but are fully functional, undergoing somatic hypermutation and isotype class switching. We have used this recombination pathway to introduce a somatically mutated variable (V) region with an unusually high affinity for the hapten p-azophenylarsonate (Ars) into the preimmune antibody repertoire. The affinity of this V region for Ars is 100-fold higher than any unmutated anti-Ars antibody previously characterized. Expression of the transgene-encoded V region did not affect many aspects of antigen-driven B cell differentiation, including somatic hypermutation, in either Ars-specific transgene- or endogenous V gene-expressing clones. Thus, the regulation of these processes appears to operate in a "global" fashion, in that the mechanisms involved are imperceptive of the relative affinities for antigen of the antibodies expressed by B cell clones participating in the immune response. In contrast, the selection of V region mutants leading to affinity maturation and memory cell formation was found to be strongly influenced by the transgenic V region, but only in clones expressing this V region. Hybridomas derived from transgene- and endogenous V region-expressing memory cells were isolated at similar frequencies from individual transgenic mice. The V regions expressed by hybridomas in both of these groups had 2- to 30-fold greater affinity for Ars than their unmutated precursors, despite the fact that the transgene-encoded precursors had 100-fold higher affinity than their endogenous counterparts. These results show that the criterion for entry into the memory compartment is established not by the affinity of a B cell's V region relative to all other V regions expressed during the response, but by the affinity of this V region relative to its unmutated precursor. Thus, the development of B cell memory is regulated in a "clone-autonomous" fashion.     

The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells

The expressed human Ig repertoire is not an equal representation of all V(H) segments present in genomic DNA. Studies have shown that a restricted set of V(H) gene segments are over-represented in Ab repertoires of fetal/neonatal and adult B cells. Additionally, this restricted set of V(H) genes is frequently expressed by autoimmune and tumor B cells. To investigate at which developmental stage a bias in the repertoire begins, we compared the V(H)3 and V(H)4 family repertoires of pre-B and immature B cells from bone marrow and mature B cells from peripheral blood of two adults. We found that the V4-34 and V4-59 gene segments of the V(H)4 family and the V3-23 gene segment of the V(H)3 family dominate the repertoires of the surface Ig-negative early pre-B as well as immature and mature B cells. Furthermore, the pattern of utilization of other V(H)3 family members suggests that certain genes that are frequently rearranged during early stages of B cell development are subsequently disfavored during later stages of B cell maturation. We conclude that the over-representation of certain V genes could arise from sequential mechanisms operating at both early and later stages of B cell development. These V(H)-mediated mechanisms might include preferential rearrangement and/or efficiency of pairing with the surrogate light chain at the surface Ig-negative, early pre-B cell stage and ligand selection at more mature, surface Ig-positive, B cell stages.     

The human immune response to red blood cell antigens as revealed by repertoire cloning

A major goal of current immunologic research is to develop specific therapeutic strategies by which the enormous diversity in immune response can be enhanced, attenuated, or eliminated, depending on the particular disease process. For nearly a century, the human immune response to red blood cell antigens has served as a paradigm for understanding the pathophysiology of autoimmune disorders and alloimmune reactions to foreign cells and tissues. Recent developments in molecular biology have facilitated the expression of immune repertoires in the form of immunoglobulin Fab fragments on the surface of filamentous bacteriophage. Such approaches have provided powerful means for producing monoclonal antibodies for research, clinical, and therapeutic applications. Our laboratory has combined these techniques with novel cell-surface selection methods to isolate extraordinarily large arrays of human antibodies to the clinically relevant red blood cell Rh(D) antigen. Our results have provided a comprehensive genetic and serologic analysis of anit-Rh(D) antibodies within single alloimmunized individuals thereby offering new insights into the development of human immune repertoires.     

Receptor editing occurs frequently during normal B cell development

Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that lambda light chain genes usually rearrange after kappa genes. This allowed us to analyze kappa loci in IgMlambda+ cells to determine how frequently in-frame kappa genes fail to suppress lambda gene rearrangements. To do this, we analyzed recombined VkappaJkappa genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the kappa locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged kappa loci, as nearly half (47%) of the RS-inactivated VkappaJkappa joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.     

Anti-adhesive signals are mediated via major histocompatibility complex class II molecules in normal and neoplastic human B cells: correlation with B cell differentiation stage

We show that major histocompatibility complex (MHC) class II molecules on B cells transit signals which regulate adhesion in a negative manner. Engagement of MHC class II molecules with antibodies results in detachment of B cells previously bound to interferon-gamma-activated human umbilical cord venous endothelial cells. This process depends on metabolic energy, active signaling and protein tyrosine kinase activity. The adhesion pathway influenced by this signaling event is neuraminidase sensitive. The anti-adhesive signaling program is activated in B cell lines with a mature phenotype, e.g. normal B cells from spleen and tonsil. In contrast, cell lines with a pre-B cell phenotype and normal B cells from peripheral blood are refractory to MHC class II-mediated regulation of adhesion. These results extend to neoplastic cells from patients with lymphoproliferative diseases representing different stages of B cell maturation. These results suggest that MHC class II-mediated signals regulate B cell adhesion in a developmentally programmed fashion; this might have implications for clinical behavior of B cell malignancies.     

Analysis of the T cell receptor repertoire in rheumatoid arthritis

BACKGROUND AND AIMS OF THE STUDY: Valve repair, where suitable, is the preferred option in patients who require mitral surgery. A number of studies have shown excellent long-term results, but most were undertaken in tertiary referral centers with a high throughput of patients. METHODS: We present our experience in 60 patients, aged 60 +/- 14 years, undergoing repair between 1984 and 1993. Most patients (83%) were in New York Heart Association (NYHA) class II or III at the time of surgery; 27% had concomitant ischemic heart disease. Almost all (98%) had posterior leaflet repair and 18% had anterior leaflet repair. Eight surgeons each performed a mean of 7.5 operations during this period. RESULTS: The 30-day mortality rate was 3.3%. There were seven late deaths. Five patients underwent reoperation for mitral regurgitation (two early, three late). At six years, 60% of patients were alive, or free of stroke or reoperation. Late follow up was obtained in 45 of 47 surviving patients: 95% were in NYHA class I or II; one-third were on anticoagulants for atrial fibrillation; 90% had mild (or less) mitral regurgitation on echocardiography. CONCLUSIONS: These data show that most patients have a very good outcome from valve repair surgery and encourage the trend towards operating earlier in the course of the disease. Adverse outcomes occurred mainly in patients who were highly symptomatic at the time of surgery. The high proportion of patients on postoperative anticoagulants underscores the importance of operating before atrial fibrillation becomes permanent.     

Cytokine repertoire of epithelial cells lining the human urinary tract

PURPOSE: To examine the cytokine profile of epithelial cells lining the human urinary tract with the aim of differentiating between the constitutive and disease-related cytokine production in these tissues. MATERIALS AND METHODS: Sections from the renal pelvis, ureter, bladder or urethra were obtained from 22 patients undergoing urinary tract surgery and were stained with monoclonal antibodies to interleukin(IL)-1beta, IL-4, IL-6, IL-8, interferon gamma (IFNgamma) and transforming growth factor beta (TGFbeta). Sections were classified according to the presence or absence of disease in the tissue. RESULTS: Epithelial cells lining the renal pelvis, ureter, bladder or urethra all stained for IL-8 and TGFbeta (100%) in disease-free tissues and sections with cancer or interstitial cystitis (IC). In contrast, staining for IL-1beta, IL-4, IL-6 and IFNgamma varied with the disease state of the patient. Epithelial IL-1beta staining was absent (0%) in sections from healthy bladder, but positive in tissues with IC or cancer-associated pathology (50 to 100%). IL-6 staining was detected in the epithelial layer of several patients with IC or cancer related pathology, but only in cells with non-epithelial morphology and not in disease-free tissues. IFNgamma and IL-4 staining were only observed in patients with IC and only in cells with non-epithelial morphology. CONCLUSIONS: The results show that epithelial cells from all parts of the urinary tract constitutively produce IL-8 and TGFbeta and suggest that the production of other cytokines varies with the disease of the patient. Constitutive cytokine production provides the basis for a rapid host response, in the defense against mucosal attack by microbes or toxic agents.