High circulating levels of soluble scavenger receptors (sCD5 and sCD6) in patients with primary Sjögren's syndrome.

OBJECTIVE: To determine the existence of circulating levels of soluble scavenger receptors (sCD5 and sCD6) in patients with primary Sjögren's syndrome (SS), and to analyse the correlation with clinical and immunological features of SS. METHODS: Ninety consecutive patients with primary SS were studied. All patients fulfilled four or more of the European diagnostic criteria for SS. sCD5 and sCD6 levels were determined using a specific enzyme-linked immunosorbent assay (ELISA) developed in our laboratory. RESULTS: Detectable levels of sCD5 were found in 39 (43%) SS patients. The mean+/-standard error values of sCD5 were 3.5+/-0.5 ng/ml for patients with SS and 1.9+/-0.1 ng/ml for healthy blood donors (P<0.001). We found higher levels of sCD5 in patients with hypocomplementaemia (6.5 vs 3.5 ng/ml, P=0.03) and cryoglobulinaemia (6.9 vs 2.6 ng/ml, P=0.001). On the other hand, detectable levels of sCD6 were found in 60 (67%) SS patients. The mean+/-standard error values of sCD6 were 25.5+/-7.8 ng/ml in SS patients and 5.27+/-1.40 ng/ml in healthy blood donors (P=0.01). When the sCD6 levels were compared according to the presence or absence of immunological features, patients with cryoglobulinaemia showed higher levels of circulating sCD6 (77.3 vs 17 ng/ml, P=0.01) than those without cryoglobulinaemia. CONCLUSION: Patients with primary SS showed higher levels of circulating sCD5 and sCD6 when compared with controls. Moreover, the existence of some immunological features (hypocomplementaemia and cryoglobulinaemia) was associated with high levels of both soluble scavenger receptors. These facts may reflect an enhanced lymphocytic activation in patients with primary SS.

CD5 signal transduction: positive or negative modulation of antigen receptor signaling.

The CD5 lymphocyte surface glycoprotein is a coreceptor involved in the modulation of antigen-specific receptor-mediated activation and differentiation signals. Although first considered a costimulatory molecule in mature peripheral T cells, recent studies of CD5-/- mice have opened the possibility that CD5 may also mediate inhibitory signals that attenuate TCR/CD3- and BCR-mediated triggering in thymocytes and a subgroup of B cells (B-1a cells), respectively. The ultimate molecular basis for these differential modulatory properties of CD5, depending on the context of lymphocyte subset and differentiation stage, are presently unknown and are an issue of current intensive investigation. Here, we review recent reports, both contradictory and complementary, focused on CD5-mediated molecular intracellular signaling events that could provide the basis for its immunomodulatory properties.

Cell surface receptors and their ligands: in vitro analysis of CD6-CD166 interactions.

CD6 is a cell surface receptor belonging to the scavenger receptor cysteine-rich (SRCR) protein superfamily (SRCRSF). It specifically binds activated leukocyte cell adhesion molecule (ALCAM, CD166), a member of the immunoglobulin (Ig) superfamily (IgSF). CD166 was among the first molecules identified as a ligand for an SRCRSF receptor, and the CD6-CD166 interaction was the first interaction characterized involving SRCRSF and IgSF proteins. We focus here on what has been learned about the specifics of the CD6-CD166 interaction from in vitro analysis. The studies are thought to provide an instructive example for the analysis of interactions between single-path transmembrane cell surface proteins. Using soluble recombinant forms, the extracellular binding domains of receptor and ligand have been identified and characterized in a variety of assay systems. Both CD6 and CD166 have been subjected to intense mutagenesis and monoclonal antibody (mAb) binding studies and residues critical for their interaction have been identified. The availability of structural prototypes of both superfamilies has made it possible to map the binding site in CD166 and, more recently, in CD6 and compare these regions to epitopes of mAbs that block, or do not block, the interaction. In addition, the molecular basis of observed cross-species receptor-ligand interactions could be rationalized. These studies illustrate the value of structural templates for the interpretation of sequence and mutagenesis analyses. Proteins 2000;40:420-428.

CD5 is A potential selecting ligand for B-cell surface immunoglobulin: a possible role in maintenance and selective expansion of normal and malignant B cells.

Although the function of CD5 on B cells is unknown, previous studies suggested that CD5 interaction with V(H) framework regions of surface immunoglobulins (Igs) may contribute to survival and expansion of B cells. Here we used B-chronic lymphocytic leukemia (B-CLL) cells and transformed B-cell lines from normal and B-CLL patients to study CD5-Ig interactions. Immobilized Ig binds and permits isolation of CD5 from lysates of CD5-expressing cell lines. Immunoglobulins or Fab fragments of different V(H) families varied in their effectiveness as inhibitors of anti-CD5 staining of CLL cells, appendix and tonsil tissue sections. Human Ig also binds to purified recombinant CD5. We show here for the first time that the unconventional Ig-CD5 interaction maps to the extracellular CD5-D2 domain whereas conventional epitopes recognized by anti-CD5 antibodies are localized in the D1 domain of CD5. We propose that interactions of VH framework regions with CD5 as a ligand may maintain, select or expand normal, autoimmune or transformed B cells and also contribute to skewing of the normal V(H) repertoire.

Relevance of individual CD5 extracellular domains on antibody recognition, glycosylation and co-mitogenic signalling.

CD5 is a type I glycoprotein which modulates T- and B-cell receptor-mediated signals and is expressed by thymocytes, mature T cells and a subset of mature B cells. The extracellular region of CD5 is composed of three scavenger receptor cysteine-rich domains (D1, D2, D3) for which only limited functional and structural data are available. Using cell transfectants expressing ectodomain-deficient CD5 molecules or CD5 immunoglobulin fusion proteins, we analysed individual CD5 domains with respect to monoclonal antibody binding specificity, glycosylation, and co-mitogenic signalling. Our results show the presence of N-linked oligosaccharides on D1 and D2, but not on D3. D1, the most amino-terminal domain, is predicted to be the most appropriately placed domain for an interaction with a ligand. This domain is recognised by a large panel of well characterised CD5 mAbs, reflecting its higher immunogenicity. In an attempt to develop mAbs with specificity for the more conserved membrane-proximal domains, we generated a unique mAb, named 83-C4, whose binding mapped to D3. Co-stimulatory studies revealed no significant differences between anti-D1 and anti-D3 mAbs. The high interspecies conservation of D3 implies a conserved role of this domain in CD5 function and the 83-C4 mAb promises to be a valuable tool in exploring this.

Interaction of recombinant and natural soluble CD5 forms with an alternative cell surface ligand.

CD5, a member of the scavenger receptor cysteine-rich (SRCR) receptor family, plays a role in the thymocyte maturation, T cell activation and T cell-antigen-presenting cell interactions. To date only CD5 ligands (CD5L) compatible with a T-B co-stimulatory role have been described (CD72, gp40-80 and IgV(H) framework region) so the existence of alternative CD5L involved in other aspects of T cell biology warrants further exploration. Here we characterize the cell binding properties of a recombinant soluble human CD5 extracellular domain glycoprotein (rsCD5). In contrast to previously characterized ligands, this molecule binds to a broadly distributed cell surface receptor expressed on monocytes, lymphocytes and various cell lines of lymphoid, myelomonocytic and epithelial origin. The cell binding of rsCD5 is divalent cation independent and inhibited by high molar concentrations of certain monosaccharides. Both human CD5 Ig fusion proteins and a natural soluble CD5 form (present in human serum and resulting from proteolytic cleavage following lymphocyte activation) reproduce the cell binding pattern of rsCD5 and block its binding in a competitive form. The involvement of the most N-terminal CD5 SRCR domains (D1 and D2) in binding is deduced from competition cell binding assays with CD5 Ig fusion proteins. These results imply a novel CD5/CD5L interaction model recalling some aspects of the interaction of CD6 with activated leukocyte cell adhesion molecule (ALCAM).

CD5 negatively regulates the T-cell antigen receptor signal transduction pathway: involvement of SH2-containing phosphotyrosine phosphatase SHP-1.

The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3zeta, ZAP-70, Syk, and phospholipase Cgammal but not the Src family tyrosine kinase p56(lck). By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.

Analysis of the tyrosine phosphorylation and calcium fluxing of human CD6 isoforms with different cytoplasmatic domains.

CD6 is a cell surface glycoprotein that functions both as a co-stimulatory and adhesion receptor on T cells. Recently we have described CD6 isoforms (CD6a, b, c, d, e) that arise via alternative splicing of exons encoding the cytoplasmic region of the molecule. CD6 becomes phosphorylated on tyrosine (Tyr) residues following stimulation through the T cell receptor (TCR) complex. Since the phosphorylation of Tyr residues renders some cell surface receptors competent for interactions with proteins of intracellular signaling pathways, we wanted to determine which region(s) and residues in the cytoplasmic domain of CD6 were important for phosphorylation on Tyr residues. We engineered and stably expressed chimeric receptors that consisted of the extracellular region of mouse CD6 and the cytoplasmic regions of either naturally occurring isoforms of human CD6, truncated proteins, or point mutants. We were able to demonstrate that of the nine Tyr residues in the cytoplasmic domain of the largest isoform CD6a, the two C-terminal Tyr residues (Tyr 629/662) are critical for the phosphorylation of CD6 following TCR cross-linking. Isoform CD6e, which is missing a region that contains two proline-rich motifs, is not phosphorylated. We further analyzed the ability of the different CD6 isoforms and truncated receptors to mobilize intracellular calcium after CD6/TCR co-ligation. All CD6 isoforms, including CD6e, as well as the truncation mutant delta 555, which is missing approximately the C-terminal half of the cytoplasmic domain, are able to increase Ca2+ influx. Taken together, these results suggest that the region of CD6 which is critical for Ca2+ mobilization is located N-terminal from amino acid 555 and is therefore different from the region located at the C terminus of CD6, which is necessary for tyrosine phosphorylation.

CD6-ligand interactions: a paradigm for SRCR domain function?

The scavenger receptor cysteine-rich (SRCR) superfamily, which includes proteins expressed by leukocytes, can be subdivided into groups A and B. Group B contains the lymphocyte cell-surface receptor CD6. This article reviews recent progress in understanding the interaction between CD6 and its ligand, activated leukocyte cell adhesion molecule (ALCAM). Analysis of the CD6-ALCAM interaction may help to understand how other SRCR domains bind to their ligands.

An infection inhibiting urinary catheter material.

Catheter associated bacteriuria is a common infection in hospitals and nursing homes. An infection inhibiting catheter material for fabricating urinary catheters is being developed. The material consists of silicone rubber elastomer compounded with chlorhexidene gluconate (CHG) matrix. The antibiotic is released in sustained fashion over at least 4 weeks. A method was established for adding CHG to silicone rubber. To protect the CHG, it is suspended in a water soluble wax that also modulates CHG release from the elastomer. It was found that CHG is randomly dispersed in the elastomer and that the primary release mechanism is by diffusion. The antibacterial activity of the material with a range of 0.1 to 5% CHG by weight was examined using in vitro zone inhibition testing. The new material demonstrated significant inhibitory activity against three pathogens tested (Escherichia coli, Proteus mirabilis and Staphylococcus epidermidis.). The release rate of CHG was measured in vitro using high performance liquid chromatography (HPLC). With 5% CHG loading, the antibiotic was released at a steady rate of approximately 8.4 mg/cm2/day for periods extending beyond 4 weeks. This new material for urinary catheters has the potential to provide protection against infection and surface colonization.

CD6 recognizes the neural adhesion molecule BEN.

CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) have been detected on various immune cells and in the brain. CD6-ligand interactions have been implicated in the regulation of T cell function. ALCAM shares the same extracellular domain organization and significant sequence homology with the chicken neural adhesion molecule BEN. Although ALCAM's CD6 binding site is only partially conserved in BEN, CD6 specifically binds BEN, albeit with approximately 10-fold lower avidity than ALCAM. Differences in binding avidity are not detected when ALCAM and BEN fusion proteins containing the full-length extracellular regions are tested. Homotypic interactions between full-length forms are likely to account for these observations. The identified cross-species interaction between CD6 and BEN suggests that CD6-ligand interactions are highly conserved.

Mutational analysis of the CD6 ligand binding domain.

CD6 belongs to the scavenger receptor cysteine-rich protein superfamily (SRCRSF), which includes a large number of cell surface proteins. The extracellular region of CD6 is composed of three SRCR domains. The membrane proximal SRCR domain of CD6 (CD6D3) specifically binds activated leukocyte cell adhesion molecule (ALCAM), a cell surface protein which is a member of the immunoglobulin superfamily (IgSF). CD6-ligand interactions have been implicated in immune cell adhesion, T cell maturation and the regulation of T cell activation. We tested 13 CD6D3 mutant proteins for binding to ALCAM and a panel of conformationally sensitive anti-CD6D3 monoclonal antibodies (mAbs). CD6D3 residues were classified according to their importance for structural integrity and ligand binding. The results were analyzed in the light of SRCR domain sequence comparison. A number of residues critical for ligand binding or important for structural integrity cluster in the C-terminal region of CD6D3 which is not conserved in other SRCR proteins.

Characterization of mouse ALCAM (CD166): the CD6-binding domain is conserved in different homologs and mediates cross-species binding.

Activated leukocyte cell adhesion molecule (ALCAM; CD166) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen CD6. Herein, we report on the isolation and characterization of cDNA clones encoding mouse ALCAM (mALCAM). Comparison of the predicted amino acid sequence of mALCAM and human ALCAM (hALCAM) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of mALCAM showed that the CD6 binding site is located in the N-terminal Ig-like domain and that mALCAM is capable of binding both human and mouse CD6. Mutagenesis studies on hALCAM suggested that residues critical for CD6 binding map to the predicted A'GFCC'C" beta-sheet of ALCAM's N-terminal binding domain. Residue differences in the N-terminal domains of mALCAM and hALCAM were analyzed with the aid of a molecular model of ALCAM. All residues critical for CD6 binding are conserved in both mALCAM and hALCAM, whereas residue differences map to the predicted BED face which is opposite the CD6 binding site on hALCAM. These findings provide a molecular rationale for the observed cross-species CD6/ALCAM interaction and the apparent inability to generate monoclonal antibodies (mAb) against the CD6 binding site. RNA blot analysis showed that mRNA transcripts encoding mALCAM are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for mALCAM was produced and by two-color immunofluorescence studies was shown to bind to both activated CD4+ and CD8+ T cells.

Identification of residues in CD6 which are critical for ligand binding.

CD6 is a member of the scavenger receptor cysteine rich protein superfamily (SRCRSF). This family includes many cell surface proteins whose three-dimensional structures and functions are presently not well understood. The extracellular region of CD6 includes 3 SRCR domains. The membrane proximal SRCR domain specifically binds the activated leukocyte cell adhesion molecule (ALCAM), a CD6 ligand belonging to the immunoglobulin superfamily. CD6-ALCAM interactions mediate immune cell adhesion and are implicated in T cell maturation and the regulation of T cell function. On the basis of SRCRSF sequence comparison, a mutagenesis analysis of the membrane proximal SRCR domain of CD6 (CD6D3) has been carried out. Fifteen mutants were characterized. Three CD6 residues were identified in a region of low sequence conservation which, when mutated, abolish ligand binding but not the binding to a panel of conformationally sensitive anti-CD6 mAbs. This study provides the first analysis of residues critical for ligand binding to a member of the SRCRSF.

Structure and chromosomal location of the human CD6 gene: detection of five human CD6 isoforms.

The CD6 protein has been shown to play important roles in T cell costimulation and adhesion. Recently, variably spliced isoforms of CD6 mRNA have been identified in both human and murine T cells. Here we report on the genomic organization of the human CD6 gene, its chromosomal localization, and the characterization of novel isoforms. Human CD6 is encoded by at least 13 exons. The amino terminal signal sequence, extracellular region, and transmembrane domain are encoded by seven exons, while the cytoplasmic domain of CD6 is encoded by six exons. Each of the three extracellular scavenger receptor cysteine-rich domains is encoded by a separate exon. Fluorescence in situ hybridization studies and screening of a chromosome-specific YAC (yeast artificial chromosome) library revealed that the gene encoding CD6 is located on chromosome 11 at 11q13 in close proximity to the gene encoding the related molecule CD5 and within 600 kb of CD20. Analysis of mRNA transcripts encoding CD6 isolated from mitogen-activated PBMC and from B cells obtained from patients with chronic lymphocytic leukemia revealed the presence of at least five different CD6 transcripts. These transcripts arise via variable splicing of exons encoding the cytoplasmic domain of CD6. The existence of these isoforms suggests that signaling through CD6 could be regulated via alternative splicing of cytoplasmic encoding exons.

Characterization, mapping and partial cDNA sequence of the 57-kD intracellular Ki-1 antigen.

A novel antigen was identified by the cross-reactivity of the anti-CD30 antibody Ki-1. This 57-kD intracellular Ki-1 antigen (Ki-1/57) is induced upon activation of leukocytes and is transported to the nuclear compartment. We describe the partial cloning and sequencing of the Ki-1/57 cDNA from a lambda gt 11-cDNA library derived from the Hodgkin-analogous cell line L540. New monoclonal antibodies were produced against the recombinant Ki-1/57 protein fragment which were used to confirm that the Ki-1/57 antigen is associated with kinase activity and is expressed in a variety of tumor cell lines and in activated but not resting leukocytes. The Ki-1/57 gene was mapped to the bands 9q22.3-31 of human chromosome 9. This is an area which appears to be associated with secondary chromosomal aberrations in acute myeloid leukemias.

Mutational analysis of the CD6 binding site in activated leukocyte cell adhesion molecule.

The interaction between CD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM) mediates adhesion of thymocytes to thymic epithelial cells. The extracellular region of ALCAM includes five Ig-like domains, and its N-terminal V-like domain specifically binds to the membraneproximal scavenger receptor cysteine-rich domain of CD6. Previously, six ALCAM residues were identified by alanine scanning mutagenesis to contribute to the interaction with CD6. All of these residues mapped to the predicted A'GFCC'C" face of ALCAM's N-terminal domain. Here we describe the results of experiments designed to further study the CD6 binding site. Other mutagenesis experiments at four previously studied sites were carried out to better understand their importance for the interaction with CD6, and different receptor binding assays were employed to compare the contribution of these and other ALCAM residues to the CD6-ligand interaction. A total of ten new ALCAM mutants were prepared, and three additional residues were identified as critical for CD6 binding. These studies have enabled us to classify ALCAM residues according to their importance for binding and to describe the CD6 binding site in some detail.

Recognition of diverse proteins by members of the immunoglobulin superfamily: delineation of the receptor binding site in the human CD6 ligand ALCAM.

The CD6-ALCAM (activated leukocyte cell adhesion molecule) interaction, which mediates thymocyte--thymic epithelial cell adhesion, is a previously unobserved type of protein--protein interaction that involves members of the scavenger receptor cysteine rich protein superfamily (SRCRSF) and the immunoglobulin superfamily (IgSF). Targeted mutagenesis of ALCAM reveals that residues which constitute the CD6 binding site cluster on the predicted A'GFCC'C" face of its N-terminal Ig domain. These results, in conjunction with recent analyses of interactions involving other IgSF members, suggest that this region in IgSF cell surface proteins is most suitable to mediate interactions with different ligands irrespective of their structure. The CD6 binding site in ALCAM is conserved across species, and nonconserved residues in ALCAM and its murine homolog map to the beta-sheet face opposite to the CD6 binding site. This provides a molecular rationale for the inability to obtain murine monoclonal antibodies against the receptor binding domain which block the CD6-ALCAM interaction.

Identification of a novel inducible cell-surface ligand of CD5 on activated lymphocytes.

CD5 is a 67-kD glycoprotein that is expressed on most T lymphocytes and on a subset of mature B cells. Although its physiologic function is unknown, several lines of evidence suggest that CD5 may play a role in the regulation of T cell activation and in T cell-antigen presenting cell interactions. Using a CD5-immunoglobulin fusion protein (CD5Rg, for receptorglobulin) we have uncovered a new CD5 ligand (CD5L) expressed on the surface of activated splenocytes. Stimulation of murine splenocytes with anti-CD3 and anti-CD28 antibodies induce transient expression of CD5L on B lymphocytes that lasts for approximately 72 h. Binding of CD5Rg to activated splenocytes is trypsin resistant and independent of divalent cations. However, it is pronase sensitive and dependent on N-linked glycosylation of CD5, since treatment of CD5Rg with PNGaseF on N-glycanase completely abrogates its ability to bind activated splenocytes. It addition to splenocytes, CD5L is expressed on activated murine T cell clones. Immunoprecipitation, antibody, and recombinant protein blocking studies indicate that CD5L is distinct from CD72, which has been proposed to be a CD5 ligand. To determine whether CD5-CD5L interaction might play a role in vivo, we tested the effect of CD5Rg in a murine model of antibody-mediated membranous glomerulonephritis. Injection of CD5Rg was found to abrogate development of the disease. Taken together, our results help identify a novel ligand of CD5 and propose a role for CD5 in the regulation of immune responses.