Differential expression of CD 180 and IgM by B-cell chronic lymphocytic leukaemia cells using mutated and unmutated immunoglobulin VH genes.

We have studied the surface expression of the Toll-like receptor family member CD 180 on cells from 78 patients with B-chronic lymphocytic leukaemia (B-CLL). B-CLL cells had variable levels of CD 180 expression, but this was always less than that expressed by normal blood B cells and was stable for 24 months. Significantly higher levels of CD 180 were expressed by B-CLL cells with mutated IGVH genes compared with those using unmutated IGVH genes. This was in contrast to the higher levels of expression of surface immunoglobulin M by B-CLL cells using unmutated, rather than mutated IGVH genes. CD 180 was functional on B-CLL cells from some of the patients, as shown by the increased expression of CD 86 following incubation in vitro with anti-CD 180. The differential expression of CD 180 amongst B-CLL patients is one more marker that may define more precisely the different biological properties of this heterogeneous disease.

Decreased expression of FcgammaRIII (CD16) by gammadelta T cells in patients with rheumatoid arthritis.

Some gammadelta T cells express a receptor for the Fc portion of immunoglobulin G (FcgammaRIII - CD16). The relevance of this Fc receptor to gammadelta T-cell function is at present unclear. Our previous studies have shown that gammadelta T cells express activation markers in patients with rheumatoid arthritis (RA). In this study we have examined the relative proportions of CD16+ gammadelta T cells in the blood and synovial fluid of these patients compared with control blood. CD16+ gammadelta T cells from RA patients were significantly reduced in synovial fluid compared with the circulation. That this was due to blocking of antibody binding to CD16 was unlikely as treatment of blood gammadelta T cells with RA synovial fluid (known to contain immune complexes) failed to alter expression of CD16. Treatment of blood gammadelta T cells with phytohaemagglutinin in vitro, resulted in a time-dependent decrease in expression of CD16, with a concomitant increase in expression of human leucocyte antigen-DR, at the single cell level. We conclude that expression of CD16 by gammadelta T cells is lost in the synovial compartment as the result of activation.

CD5 B cells and B-cell malignancies.

Over the past year, progress has been made in understanding of the physiology and disease associations of CD5+ (B1) B cells, although their exact role in pathogenesis remains unclear. Earlier studies on the negative function of CD5 within the B-cell receptor complex have been substantiated, and it seems likely that soon the signaling pathways used by this coreceptor will be elucidated. Progress in diagnosis, physiology, and etiopathogenesis of CD5+ malignancies has been made, particularly in B-cell chronic lymphocytic leukemia. The low-level expression of surface immunoglobulin has been explained by the mutations that occur in the associated CD79b. Two new potential tumor-suppressor genes have been identified in the hot spot of chromosome 13q, which provides an exciting step forward in understanding of the etiopathogenesis of some B-cell chronic lymphocytic leukemia. Activated signal transducers for activation of transcription factors molecules have been shown to be phosphorylated on different amino acids in B1 and chronic lymphocytic leukemia tumors, although the significance of this is, as yet, unclear. Finally, aberrant expression of CD40L by chronic lymphocytic leukemia T cells may contribute to the immunodeficiency that develops in these patients.

The binding of some human antiendothelial cell antibodies induces endothelial cell apoptosis.

The pathogenic role of antiendothelial cell antibodies (AECA) remains unclear. They are frequently associated with antibodies to anionic phospholipids (PL), such as phosphatidylserine (PS), which is difficult to reconcile with the distribution of PL molecular species within the plasma membrane. Since it is already known that PS is transferred to the outer face of the membrane as a preclude to apoptosis, the possibility exists that apoptosis is initiated by AECA. AECA-positive/anti-PL antibody-negative sera from eight patients with systemic sclerosis (SS) and 21 control patients were evaluated. Endothelial cells (EC) were incubated with AECA and the exposure of PS was established through the binding of annexin V. Hypoploid cell enumeration, DNA fragmentation, and optical and ultrastructural analyses of EC were used to confirm apoptosis. Incubation of EC with AECA derived from six of eight patients with SS led to the expression of PS on the surface of the cells. This phenomenon was significantly more frequent in SS (P < 0.04) than in control diseases. The redistribution of plasma membrane PS preceded other events associated with apoptosis: hypoploidy, DNA fragmentation, and morphology characteristic for apoptosis. Apoptosis-inducing AECA did not recognize the Fas receptor. We conclude that AECA may be pathogenic by inducing apoptosis.

Antiendothelial cell antibodies: useful markers of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SS) encompasses a wide spectrum of clinical presentations. Antiendothelial cell antibodies (AECA) in patients with primary Raynaud's phenomenon (PRP), limited SS (lSSc), or diffuse SS (dSSc) may help to determine the long-term prognosis of the disease. METHODS: Twenty-seven normal controls, 13 patients with PRP, 36 with lSSc, and 31 with dSSc were included in the study. Sera were examined for the presence of AECA, using a cellular enzyme-linked immunosorbent assay (ELISA). Angiotensin-converting enzyme (ACE) activity, plasma von Willebrand factor antigen (vWfAg), and thrombomodulin (Tm) concentrations were also evaluated. The medical records of 50 of the lSSc and dSSc patients were reviewed and the organ system involvement noted. RESULTS: Antiendothelial cell antibodies were present in 3 patients with PRP, 16 patients with lSSc, and 26 patients with dSSc. These autoantibodies were mainly of the IgG isotype. There was no difference in ACE activity between patients and controls. In contrast, vWfAg and Tm concentrations were higher in patients with PRP relative to controls, and higher in patients with lSSc compared with those with PRP. The presence of AECA was associated with digital scars and ulcers (P < 0.004 and P < 0.003, respectively), severe RP (P < 0.01), grade 3 tortuosity of vessels (P < 0.0004), and lung involvement (P < 0.02). CONCLUSION: The significant trend for AECA to increase with disease severity across the three groups of patients studies suggests that the AECA test can identify subsets of SSc with differing prognoses.

CD5+B cells: differential capping and modulation of IgM and CD5.

CD5 is associated with the B-cell antigen receptor (BcR) complex. As an approach to understanding its role in B-cell function, the authors investigated the capping and modulation of CD5 and surface IgM (sIgM). Tonsillar B cells were treated with anti-IgM or anti-CD5 antibodies, capping examined after 1 h (by fluorescence microscopy) and modulation after 24 h (by flow cytometry). CD5 co-capped and co-modulated with sIgM. Of various drugs tested, only the protein tyrosine kinase inhibitor (genistein) had any effect on capping and co-capping. Capping of sIgM (and co-capping of CD5) but not capping of CD5 (or co-capping of sIgM) was inhibited by genistein. None of the other drugs affecting PKC or cytoskeletal structures (colchicine and cytochalasin D) had any effect. However, the PKC inhibitors, staurosporine and H-7, inhibited the modulation of sIgM by anti-IgM but not CD5 by anti-CD5. In contrast, PKC activators, PMA and mezerein, inhibited modulation of CD5 by anti-CD5 but not sIgM by anti-IgM. This suggests that direct ligation of CD5 utilizes different signalling pathways compared with sIgM. It seems likely that in CD5+ cells, interaction of CD5 with its ligand CD72 modulates signals transmitted through the BcR.

The majority of Fc gamma RIII-positive gamma delta T cells do not express HLA-DR in patients with primary Sjögren's syndrome.

The percentages of circulating gamma delta T cells and the proportions of these expressing Fc gamma RIII (CD16) or HLA-DR in patients with primary Sjögren's syndrome (pSS) and controls were determined using monoclonal antibodies and flow cytometry. There was no significant difference in the percentages of gamma delta T cells in the pSS patients compared with controls. There was, however, a significant increase in the proportions of both CD16+ and HLA-DR+ gamma delta T cells in pSS patients. A 3-colour immunofluorescence technique demonstrated that these two markers were mutually exclusive and therefore may identify either subpopulations of gamma delta T cells or different stages of the activation process.

Selective expression of V beta families by T cells in the blood and salivary gland infiltrate of patients with primary Sjögren's syndrome.

OBJECTIVE: The T cell infiltration of the salivary gland of patients with Sjögren's syndrome (SS) has been implicated in the pathogenic process of the disease. We examined the representation of V beta subsets in the blood and salivary tissue of patients with SS. METHODS: Circulating T cells from 12 patients and paired samples of blood and labial salivary glands obtained from 8 patients were studied. A panel of monoclonal antibodies directed against the variable region of the T cell receptor was used to enumerate the cells expressing V beta families in the peripheral blood by flow cytometry, and in tissue sections by immunofluorescence. RESULTS: We found an increase of cells bearing V beta 2 family gene products in the circulation, and an increase in both V beta 2 and V beta 8 in the salivary gland infiltrate of patients with SS. No significant difference was noted between the 5 DR3+ patients and 7 DR3- patients studied with regard to the V beta families seen. CONCLUSION: Our data are consistent with a role for specific T cell families in the pathogenesis of SS.

The genetic basis of human VH4 gene family-associated cross-reactive idiotype expression in CD5+ and CD5- cord blood B-lymphocyte clones.

In a recent study we have observed a high frequency expression of cross-reactive idiotypes encoded by genes from the relatively small VH4 family of immunoglobulin heavy chain genes in cord blood B-lymphocyte lines. Furthermore, we have demonstrated a selective pattern of expression of two VH4-associated cross-reactive idiotype (CRI) in B-lymphocyte lines established from CD5+ and CD5- cord blood B-lymphocytes. There was a restricted expression of one CRI marker recognized by the 9G4 monoclonal antibody in lines established from CD5+ B-lymphocytes but not in those established from the CD5- population. In the current study we examine the molecular basis for the selective pattern of CRI expression. Nucleotide-sequence analysis of functional immunoglobulin heavy chain (IgH) gene rearrangements in three CD5+ lines expressing the CRI recognised by 9G4 reveal that all use a single gene from the VH4 family, the V4.21 gene. However, all three lines have distinct third complementarity determining regions (CDR3) implying different clonal origins. In contrast, four cord blood cell lines (two established from CD5+ B-lymphocytes) expressing the CRI recognized by MoAb Lc1 have functional IgH gene rearrangements involving two different genes from the VH4 family, the V71-4, and V2-1 genes. Antigen specificity analysis reveals that all three 9G4-reactive lines produce antibodies that react with the I and/or i red blood cell carbohydrate antigens. These data suggest that the distinction in VH4 gene use in CD5+ B-lymphocytes in cord blood results from a selection process in vivo that shapes the repertoire of CD5+ B-lymphocytes. This study extends recent observations that the monoclonal anti-CRI antibodies 9G4 and Lc1 are markers of two distinct subgroups of proteins encoded by two subsets of genes within the VH4 family. Furthermore, it appears that amino acid residues in framework region one and complementarity determining region two are critical for the expression of the cross reactive idiotypes and the serological distinction between the two subgroups of proteins.

Specificity and idiotope expression of IgM produced by CD5+ and CD5- cord blood B-cell clones.

Epstein-Barr virus (EBV)-immortalized monoclonal B-cell lines were established from CD5+ and CD5- cord-blood B cells. IgM from many of both CD5+ and CD5- clones reacted with IgG-Fc, ssDNA, and a variety of other autoantigens. More CD5+ B cells that used light chains of the kappa isotype reacted with IgG-Fc and ssDNA than kappa-bearing CD5- B cells. Because many of the clones reacted with IgG-Fc, they were analyzed for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor and cold agglutinin paraproteins using murine antibodies (mAb) recognizing V kappa and VH subgroup-associated determinants. Expression of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17.109 mAb was expressed at significantly higher frequency (32%; p less than 0.05) and IgM antibodies derived from the CD5+ compared with the CD5- clones (5%). Both CD5+ and CD5- clones expressed the RF paraprotein-associated idiotope recognized by G8 mAb to the same extent. Similar results were obtained using binding to SpA as a marker of VH III family usage. Furthermore, no differences in frequency of expression of RF paraprotein-associated idiotopes recognized by B6 and/or D12, and characteristic of some antibodies using VH III family genes, were found between the CD5+ and CD5- populations. Although a higher than expected frequency of VH IV-gene expression was demonstrated (around 30%) in both CD5+ and CD5- cells, there were differences in expression of CRI recognized by mAb Lc1 and R2.1A2 with specificities for two VH IV subfamilies. While some CD5+ and CD5- clones were identified in which their IgM reacted with mAb Lc1, only CD5+ clones were recognized by another mAb R2.1A2. Analysis of the relationships between antigen specificities and V kappa- and VH-family gene usage indicated that auto- or polyreactivity was not associated with V kappa III nor any particular VH family. The higher frequency of the V kappa IIIb sub-subgroup-associated idiotope recognized by 17-109 in the CD5+ clones and the association of CD5+ B cells with the VH IV subfamily recognized by mAb R2.1A2 and 9G4 may suggest that CD5+ B cells in cord blood are expanded as a result of recruitment within the fetal environment.

Repertoire of CD5+ and CD5- cord blood B cells: specificity and expression of VH I and VH III associated idiotopes.

Epstein-Barr (EBV)-immortalized B cell clones were established from CD5+ and CD5- cord blood B cells separated by flow cytometry. We have previously shown that IgM from many of the clones was polyreactive, exhibiting reactivity with a number of autoantigens. In this study, IgM produced by the clones was analysed by MoAb for the expression of cross-reactive idiotypes (CRI) associated with rheumatoid factor paraproteins and from defined VH and V kappa subgroups of immunoglobulin heavy and light chains. IgM produced by clones established from CD5+ and CD5- B cells expressed the VH I associated idiotope G8. Furthermore, IgM produced by both sets of clones exhibited a similar frequency of VH III heavy chain subgroup expression, as determined by reactivity with staphylococcal protein A (SpA) and VH III-associated CRI expression (B6 and/or D12). In contrast, expression of the V kappa III-associated 17.109 CRI was significantly higher in IgM antibodies produced by clones established from CD5+ compared with the CD5- clones (32 versus 5%: P less than 0.05). Analysis of the VH and VL subgroup expression by IgM produced by the CD5+ and CD5- cord blood clones, and their autoantigen reactivity profile did not reveal restriction or selection within CD5+ and CD5- populations. However, our data suggest that differences may exist in the expression of certain germ-line genes between CD5+ and CD5- cord blood B cells and might indicate an expansion of CD5+ B cells within the fetal environment.

Auto- and polyreactivity of IgM from CD5+ and CD5- cord blood B cells.

The presence of the CD5 (67 kDa) molecule on the surface of B cells has been considered a marker for cells producing auto- and polyreactive antibodies. Cord blood B lymphocytes (rich in CD5+ B cells) have been sorted into CD5 positive and negative populations by flow cytometry using monoclonal antibodies to CD20 and CD5. Clones of these populations were obtained by immortalization with Epstein-Barr virus. Clones derived from both CD5+ and CD5- B cells produced IgM which was auto- and polyreactive with a higher frequency of these specificities in the CD5+ population. These data indicate that expression of surface CD5 on cord blood B cells is not a definitive marker of an auto/polyreactive population.

The antibody repertoire of early human B cells. III. Expression of cross-reactive idiotopes characteristic of certain rheumatoid factors and identifying V kappa III, VHI, and VHIII gene family products.

Our previous studies have shown that a high frequency of Epstein-Barr virus (EBV)-immortalized cord blood (CB) and fetal liver (FL) clones produce IgM antibodies which display extensive autoreactivity for IgG Fc (rheumatoid factor, RF). To investigate further the repertoire of these early B cells, we have examined the expression of CRI associated with RF paraproteins in relation to antibody specificity and polyreactivity. CRI were detected by ELISA and/or flow cytometry using a panel of well-characterized monoclonal antibodies defining idiotopes associated with particular V kappa and VH gene family products and raised against Fc-specific paraproteins. Many of the CRI were expressed by these clones, suggesting that they may be markers of early B cells. The presence of the CRI was not always associated with Fc specificity. Three of eight CB/FL clones expressed the V kappa III subgroup of light chains, and two of these expressed the V kappa III sub-subgroup associated CRI, 17-109. These two clones reacted with IgG Fc, and one also bound to single-stranded DNA. The VHIII-associated idiotope D12 was expressed on IgM from 4 out of 9 FL and 5 out of 12 CB clones. D12 and B6 (also a VHIII-associated CRI) were coexpressed in 4 out of 5 CB clones but not in the four FL clones. Seven out of nine clones expressing these idiotopes were polyreactive, and five had Fc-binding activity. Three of the 12 CB clones expressed the VHI-associated conformational idiotope G8. One of 20 CLL clones expressed both B6 and D12, and another expressed both 17-109 and the VHI-associated G6 and G8 idiotopes. Taken together, these data provide evidence for the frequent usage, in early B cells, of V kappa subgroups and VH-associated idiotopes of RF paraproteins. The expression of these CRI was not a prerequisite for binding to IgG Fc, but there was a frequent association of these idiotopes with it. Differences in expression of CRI between CLL and early B-cell clones may suggest differences in the pattern of VH usage between these subsets of B cells.

Monoclonal process in primary Sjögren's syndrome and cross-reactive idiotype associated with rheumatoid factor.

Monoclonal or oligoclonal B cell products have been described in the sera and urine of patients with primary Sjögren's syndrome (PSS). In addition, monoclonal expansion of plasma cells has been found in the exocrine glands of PSS patients with circulating monoclonal B cell products. The goal of this study was to raise an anti-idiotype to a cryoprecipitable monoclonal IgM kappa rheumatoid factor (RF) from a PSS patient. Using the F(ab')2 fragments of the rabbit IgG anti-idiotype, an idiotype-specific ELISA was developed and sera from 32 patients with PSS (13 with monoclonal IgM kappa), 33 with rheumatoid arthritis, three with rheumatoid arthritis + Sjögren's syndrome (SS), 30 with systemic lupus erythematosus, six with Waldenström's macroglobulinaemia, and 20 healthy controls were tested. The idiotype was primarily found in PSS patients with circulating monoclonal IgM kappa and more often in those who had a ratio of kappa: lambda intracytoplasmically positive plasma cells greater than 3:1 in the lymphocytic infiltrates of minor salivary glands, and systemic manifestations. The idiotype was also found in PSS and rheumatoid arthritis patients without circulating monoclonal cryoglobulins as well as in two of the six patients with Waldenstrom's macroglobulinaemia. Our results suggest that the monoclonal process observed in PSS could involve restricted idiotypic clones that are susceptible to malignant transformation.

Sjögren's syndrome: an oligo-monoclonal B cell process.

Primary Sjögren's syndrome (1 degrees SS) has been considered as a privileged model for the study of autoimmunity and B-cell neoplasia. Previous and recently accumulated information have reinforced this view. The given higher incidence of non-Hodgkin's lymphoma (NHL) in 1 degree SS patients, the presence of circulating monoclonal immunoglobulins, the detection of uniform immunoglobulin gene rearrangements and monoclonal B-cell expansions in the lymphocytic infiltrates of salivary gland, the increased levels of circulating CD5 positive B-cells and the association of these cells with the presence of monoclonal immunoglobulins from 1 degree SS, and finally the finding of shared cross reactive idiotypes on monoclonal immunoglobulins from 1 degree SS and B-cell malignancies, all provide evidence of common pathogenetic links between benign and malignant lymphoproliferation.

The antibody repertoire of early human B cells. II: Expression of anti-DNA-related idiotypes.

Cord blood B cells were immortalized in vitro with Epstein-Barr virus (EBV). Supernatants containing greater than 500 ng/ml IgM from clones/lines were tested for expression of anti-DNA-associated 16/6 and PR4 idiotypes (id) by ELISA. Four of 70 lines, but no clones, were positive for 16/6 id and none expressed the PR4 id. The presence of 16/6 id on four cell lines was associated with specificity for ssDNA, cardiolipin and Fc of IgG. No association was seen with binding to the K30 polysaccharide of Klebsiella. One clone binding this antigen also had anti-ssDNA, anti-Fc and anti-cardiolipin activity but did not express 16/6 id. Our data support the germ-line nature of the 16/6 id and are consistent with the notion that IgM autoantibody-producing B cells use VH genes which are part of the normal B cell repertoire.

Current concepts in immune derangement due to undernutrition.

Protein-calorie malnutrition (PCM) adversely affects more or less all immune competent cells. Nonspecific immunity is impaired, particularly adherence and chemotaxis of phagocytes, although the responsiveness of circulating cells may not be the same as that of noncirculating cells. PCM results in numerical and functional impairment in lymphocytes. PCM markedly affects IgG class antibodies which have the highest affinity when directed against T-dependent antigens. These impairments are interrelated, since cooperation between T-helper cell and B-cells is depressed, and the antigen presentation to T-helper cells by macrophages is deficient.

Immune complexes and autoantibodies in patients with giant cell arteritis and their relationship with autologous rosette-forming cells.

Serologic studies and lymphocyte analysis were carried out in 29 patients with giant cell arteritis (GCA). IgA-containing circulating immune complexes (CIC) were detected in 16 GCA patients with or without polymyalgia rheumatica (55%). A significant difference was demonstrated in autologous rosette-forming cells between the patients as a whole, and matched controls (8.6 +/- 2.0 vs 11.6 +/- 2.4, p less than 0.001), and also between patients with and patients without CIC (7.9 +/- 1.6 vs 9.4 +/- 2.0, p less than 0.001).