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.

Identification of a novel single-nucleotide polymorphism (Val554Ile) and definition of eight common alleles for human IL4RA exon 11.

The interleukin (IL)-4 receptor alpha chain gene (IL4RA) is a polymorphic gene which is reportedly involved in the development of atopy. Of the 14 single-nucleotide polymorphisms (SNP) reported to date in the coding region of IL4RA, 11 are positioned to exon 11. This big exon encodes more than two thirds of the mature protein, including most of the cytoplasmic region. Here we report the identification of a new IL4RA SNP at the first nucleotide of codon 554 (GTA --> ATA) in exon 11, leading to an amino acid substitution from Val to Ile (V554I). Furthermore, we present complete nucleotide sequence data for eight common alleles resulting from combinations of 9 out of the 12 SNP at IL4RA exon 11. Homo- or heterozygous combinations of these eight alleles accounted for all the IL4RA exon 11 genotypes found in Caucasian individuals from our geographical area.

Residues Y429 and Y463 of the human CD5 are targeted by protein tyrosine kinases.

The human CD5 lymphocyte cell surface co-receptor modulates activation and differentiation responses mediated by the antigen-specific receptor of T and B cells. CD5 is phosphorylated following lymphocyte activation; however, the exact sites and kinases involved are yet to be determined. Jurkat T cell transfectants expressing tyrosine-mutated CD5 molecules have been used to show that residues Y429 and Y463 are targeted in vivo by protein tyrosine kinases following cell stimulation with anti-CD3 mAb or pervanadate. This is in agreement with data from direct in vitro kinase assays using purified recombinant Lck and Fyn protein tyrosine kinases. The analysis of Lck- and CD3-deficient Jurkat cells shows that tyrosine phosphorylation of CD5 requires Lck activity. We propose that T cell activation mediates CD5 tyrosine phosphorylation at residues Y429 and Y463 mainly through the activation of Lck.

Role of two conserved cytoplasmic threonine residues (T410 and T412) in CD5 signaling.

CD5 is a transmembrane coreceptor that modulates activation and differentiation signals mediated by the Ag-specific receptor present on both T and B1a lymphocytes. CD5 lacks intrinsic catalytic activity, and its immunomodulatory properties result from intracellular interactions mediated by the CD5 cytoplasmic tail. The nature of these interactions is currently a matter of investigation. Here, we present a selective mutagenesis analysis of two conserved threonine residues (T410 and T412) located at the membrane-proximal cytoplasmic region of CD5. These residues are contained within consensus phosphorylation motifs for protein kinase C and are shown here to be critical for in vivo protein kinase C-mediated phosphorylation of CD5. Functional studies revealed that the integrity of T410 and T412 is also critical for CD5-mediated phosphatidylcholine-specific phospholipase C (PC-PLC) activation and phorbol ester-mediated inhibition of Ab-induced internalization of CD5. These results strongly argue in favor of a role for T410 and T412 in the signaling mediated by CD5.

Genomic organization of the human CD5 gene.

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.

Identification of a novel HLA-Cw*07 variant (Cw*0714) in a German Caucasian family.

We report herein the identification of a new HLA-Cw*07 allele in two members of a German Caucasian family. This novel allele, designated as Cw*0714, differs from Cw*07011 and Cw*0706 by two nucleotide changes: one at codon 66 (AAC-->AAG) in the exon 2, leading to an amino acid change from Asn to Lys; and another silent substitution at codon 99 (TAT-->TAC) in the exon 3. The latest substitution (T-->C at the third position of codon 99) was not seen in any of the HLA-Cw*07 alleles reported so far, thus being characteristic to the new HLA-Cw*0714 allele.

Identification of a natural soluble form of human CD5.

CD5 is a 67 kDa type I glycoprotein which belongs to the Scavenger Receptor Cysteine-Rich (SRCR) family of receptors. This family includes either cell-surface (e.g. CD6) or secreted (e.g. Spalpha) proteins implicated in the development of the immune system and the regulation of immune responses. In this study, we purified and characterised a circulating natural soluble CD5 form (nsCD5) which is indistinguishable (in apparent molecular mass, glycosylation pattern, and antibody reactivity) from a recombinant soluble CD5 form (rsCD5) composed of the three extracellular SCRC domains. The nsCD5 is a N-glycosylated 52 kDa molecule present in normal human serum and in supernatants of in vitro phorbol ester- and CD3-stimulated peripheral blood mononuclear cells. The nsCD5 concentration in sera from healthy donors is relatively low (median 1.75 ng/ml, rn=166) and is similar to that found in sera from patients suffering of various autoimmune (systemic lupus erythematosus, primary Sjogren syndrome, rheumatoid arthritis) and non-autoimmune (chronic renal failure, B-cell chronic lymphocytic leukemia) disorders. In vitro experiments indicate that nsCD5 is released by proteolytic cleavage of the membrane form. These results represent the first evidence of proteolytic release of a transmembrane SRCR family member following cell activation.

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).

Signaling through CD5 involves acidic sphingomyelinase, protein kinase C-zeta, mitogen-activated protein kinase kinase, and c-Jun NH2-terminal kinase.

The CD5 lymphocyte surface glycoprotein is a coreceptor involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals. The molecular basis for its modulatory properties is not yet well understood. In the present study we describe early biochemical events triggered by CD5 stimulation, which include the phosphatidylcholine-specific phospholipase C (PC-PLC)-dependent activation of acidic sphingomyelinase (A-SMase) in normal and lymphoblastoid T and B cells. The functional coupling of PC-PLC and A-SMase is demonstrated by the abrogation of A-SMase activation by 1) xanthogenate tricyclodecan-9-yl (D609), a selective inhibitor of PC-PLC, and 2) replacement of several C-terminal serine residues (S458, S459, and S461) present in the cytoplasmic tail of CD5 that are known to be critical for PC-PLC activation. Additionally, we demonstrate that activation of protein kinase C-zeta (PKC-zeta) and members of the mitogen-activated protein kinase (MAPK) cascade (MAPK kinase and c-Jun NH2-terminal kinase), but not the NF-kappaB, are downstream events of the CD5 signaling pathway. A-SMase, PKC-zeta, and MAPK family members are key mediators of cell responses as diverse as proliferation, differentiation, and growth arrest and may contribute to CD5-mediated modulation of TCR or BCR signaling.

Human CD5 signaling and constitutive phosphorylation of C-terminal serine residues by casein kinase II.

CD5 is a lymphocyte surface glycoprotein with a long cytoplasmic domain suitable for phosphorylation and signal transduction, which is involved in the modulation of Ag-specific receptor-mediated activation and differentiation signals. In this study, we use Jurkat T cell transfectants of CD5 cytoplasmic tail mutants to reveal phosphorylation sites relevant to signal transduction. Our results show that casein kinase II (CKII) is responsible for the constitutive phosphorylation of CD5 molecules at a cluster of three serine residues located at the extreme C terminus (S458, S459, and S461). Furthermore, the yeast two-hybrid system demonstrates the specific association between the C-terminal regions of the CD5 cytoplasmic tail and the regulatory beta subunit of CKII. We demonstrate that CKII associates with and phosphorylates the C-terminal region of CD5, a conserved domain known to be relevant for the generation of second lipid messengers, and thereby enables at least one component of its signaling function.

Isolation of two CD50 (ICAM-3)-negative Jurkat T-cell clones and their application for analysis of CD50 function.

The leukocyte differentiation antigen CD50 (intercellular adhesion molecule-3, ICAM-3), mediates cell-cell adhesion through its ligand LFA-1 and is a transducting receptor molecule during T-cell activation. Since CD50 homologues in other species have not yet been identified, the role of this molecule can only be analyzed in human cell models. Thus, to better study CD50 function in T cells, we have obtained two CD50-negative T-cell clones, named CAMY.1 and CAMY.2. These clones were derived from the Jurkat T-cell variant PPL.1. Data from analysis of protein expression, specific mRNA content and calcium mobilization assays have confirmed the absence of functional CD50 molecules on these two clones. Thus, CAMY.1 and CAMY.2 show no CD50 expression by phenotypical and immunoprecipitation analysis. CD50-specific mRNA content is undetectable by Northern blot analysis in these clones and, only, when RT-PCR was performed could specific mRNA be detected. Additionally, CD50 cross-linking on theses clones shows no increase in intracellular calcium. Transfection of CD50 cDNA on CAMY cells restores not only CD50 surface expression, but its functional ability to induce calcium mobilization, CD69 upregulation and cell morphological changes. The CAMY.1 and CAMY.2 clones provide useful model systems to analyze CD50 function in T cells.

The cytoplasmic domain of CD5 mediates both TCR/CD3-dependent and -independent diacylglycerol production.

CD5 is a 67-kDa surface glycoprotein found in association with the Ag receptor complex on both T and B lymphocytes. CD5 modulates Ag receptor-mediated immune responses, but the molecular mechanisms of its action remain unclear. In this respect, the assessment of the relative and unique contribution of CD5 in cell signaling events is a crucial point. We have used Jurkat variants and anti-CD5 mAbs to show that the CD5 signaling pathway is distinct from that used by the TCR/CD3 complex. We hereby identify two independent mechanisms of CD5-mediated diacylglycerol release by virtue of their different kinetics: 1) an early and transient diacylglycerol increase that results from the activation of a phosphatidylcholine-specific phospholipase C, and 2) a late and sustained increase that requires de novo phospholipid synthesis. Studies performed on a TCR/CD3-deficient Jurkat cell variant indicate that only the CD5-mediated phosphatidylcholine-specific phospholipase C activation is dependent on TCR/CD3 expression. Mutational analyses of CD5 demonstrate that both mechanisms are dependent on the integrity of the CD5 distal cytoplasmic region. Our results show that CD5 is a signaling molecule per se that uses mechanisms resembling those used by some cytokine receptors (such as IL-1 or TNF receptors) to modulate lymphocyte activation.

CD50 (intercellular adhesion molecule 3) stimulation induces calcium mobilization and tyrosine phosphorylation through p59fyn and p56lck in Jurkat T cell line.

The leukocyte differentiation antigen, CD50, has been recently identified as the intercellular adhesion molecule 3 (ICAM-3), the third counter-receptor of leukocyte function-associated antigen 1 (LFA-1). This molecule seems to be specially involved in the adhesion events of the initial phases of the immune response. To characterize the role of CD50 in leukocyte interactions, the different molecular events induced after cross-linking of CD50 on T cell-derived Jurkat cell line have been analyzed. When cells were incubated with anti-CD50 mAbs and cross-linked with polyclonal goat anti-mouse immunoglobulins, a rise in intracellular calcium concentration ([Ca2+]i) was observed. This increase in [Ca2+]i was mainly due to the uptake of extracellular Ca2+. This Ca2+ flux involved tyrosine phosphorylations and was further increased by CD3 costimulation. These data, together with those obtained by phosphotyrosine (P-Tyr) immunoprecipitation and in vitro kinase assays, suggested the involvement of protein-tyrosine kinases (PTK) in CD50 transduction pathways. By using specific antisera, the presence of p56lck and p59fyn protein tyrosine kinases (PTK) was clearly demonstrated in the CD50 immunoprecipitates. These findings suggest that the interaction of CD50 with its natural ligand (LFA-1) may result in T lymphocyte activation events, in which CD50 could play a very active role after antigen triggering.

Association of an activation inducible serine kinase activity with CD5.

We show the association of a protein kinase activity with CD5 immunoprecipitates under different detergent conditions (1% digitonin, 1% Triton X-100). This association can be observed in all CD5+ cell types tested (PBMC, thymocytes, B cells from chronic lymphocytic leukemia, and some lymphoblastoid T cell lines as Jurkat, Molt-4, 8402). Phosphoaminoacid analysis of the in vitro phosphorylated proteins and Western blot analysis of the immunoprecipitates with an antiphosphotyrosine mAb show that, in contrast with other lymphocyte receptors (CD3, CD4, IL-2R), CD5 coimmunoprecipitates a serine kinase activity. Our results show also that preactivation of cells through the CD3/TCR complex induces a rapid (detectable in 1-3 min) and transient (returns to basal levels after 10-15 min) increase in the kinase activity associated with CD5 immunoprecipitates. This CD3-induced increase in CD5-associated kinase activity correlates with an increase in CD5 phosphorylation. Furthermore, activation with soluble anti-CD5 mAb induces also an increase in the kinase activity associated with this receptor. In contrast with the increase observed after activation with CD3, after activation with CD5 the increase in the kinase activity peaks after 10 min and is maintained for 1 h. These different kinetics suggest that there may exist different mechanisms that regulate this phenomenon.

Different mechanisms regulate the monoclonal antibody-induced modulation of CD2, CD3, and CD5 in human lymphocytes.

The CD2, CD3, and CD5 antigens are down-modulated from the cell surface of peripheral blood mononuclear cells after a 24-hr incubation with specific monoclonal antibodies (mAb). Here we show that active (phorbol myristate acetate, phorbol dibutyrate acetate, and mezerein) but not inactive (4 beta-phorbol) tumor-promoting agents inhibit the mAb-induced modulation of CD2 and CD5, but not CD3, without concomitant changes in the surface distribution of these antigens (such as capping). This inhibitory effect is not protein synthesis dependent and is reversed by protein kinase C inhibitors (staurosporine and H-7). The use of cytoskeleton-disrupting agents shows the existence of different cytoskeletal interactions driving the mAb-induced modulation of CD2 and CD5 with respect to CD3. Treatment with cytochalasin D (an agent that inhibits microfilament polymerization) but not colchicine (an agent that inhibits microtubule polymerization) reproduced the effect of TPA on the mAb-induced modulation of CD2, CD3, and CD5. Our results indicate that the mAb-induced modulation of CD2 and CD5 is dependent on microfilament (namely actin) polymerization and PKC activation, while the modulation of CD3 is not.

Isolation and characterisation of a CDw50 negative Jurkat T-cell line variant (PPL.1).

PPL.1, a Jurkat cell line variant deficient in CDw50 surface expression, has been selected by fluorescence-activated cell sorting and expanded in cell culture. We have studied the expression of several leukocyte surface markers (CD2, CD3, CD4, CD8, CD7, CD26, CD25, CD14, CD18, kCD20, CD43, CD45, CD45R, CD71 and HLA class I and II) and we find no differences in their expression between PPL.1 and its parental Jurkat cell line. Immunoprecipitation analysis of metabolically labelled PPL.1 cells ([35S]-cysteine plus [35S]-methionine) fails to detect the presence of a preformed cytoplasmic pool of CDw50 molecules. The deficient CDw50 expression on PPL.1 cells is stable after several weeks of continuous culture and even after exposure of cells to several lymphocyte activating agents (PGE2, PHA, Con A, calcium ionophore A23187 and human recombinant IFN-gamma). No karyotype changes responsible for such phenotype deficiency are found. PPL.1 cells are as efficient as wild-type Jurkat or K562 cells, when used as targets in cytotoxicity assays with fresh or PHA-stimulated peripheral blood lymphocytes. No blocking effects of CDw50-specific mAb are observed in such assay. These results are consistent with the fact that CDw50 is not involved in alloreactive T-cell-specific cytotoxicity. They also suggest that this antigen is implicated only on a very specialized type of cell-cell interactions.

Intracellular events involved in CD5-induced human T cell activation and proliferation.

In this report we describe a novel pathway of human T cell activation and proliferation involving the CD5 surface Ag. The CD5-specific Cris1 mAb induces by itself monocyte-dependent proliferation of PBMC. Among a panel of CD5-specific mAb (Leu1, OKT1, LO-CD5a, F101-1C5, and F145GF3), only the F145GF3 mAb shared this property with Cris1. The analysis of the biochemical pathway involved in this activation showed the lack of detectable hydrolysis of inositol phosphates or early increments in the intracellular cytosolic calcium concentration, after triggering cells with the mitogenic CD5 mAb. However, stimulation with CD5 induces activation of protein kinase C, as measured by phosphorylation of a specific peptide substrate (peptide GS), which can be inhibited by a pseudosubstrate peptide inhibitor. Stimulation with CD5 mAb induces also tyrosine kinase activity, with a substrate pattern that differs from that induced after triggering lymphocytes through the TCR-CD3 complex. On the other hand the IL-2/IL-2R pathway seems involved in the CD5-mediated proliferation of PBMC because anti-IL-2R-specific mAb inhibits CD5-induced proliferation, and stimulation with mitogenic CD5 mAb induces production of IL-2 and expression of IL-2R alpha and beta chains. Therefore, the triggering of the CD5 Ag can induce IL-2- and monocyte-dependent human T cell proliferation by a biochemical pathway that differs, at least in the first stages, from the one that mediates TCR-CD3 complex-induced T cell activation.

Effect of protein kinase C activators on the phosphorylation and the surface expression of the CDw50 leukocyte antigen.

The CDw50 antigen is a constitutively non-phosphorylated leukocyte surface molecule which becomes highly phosphorylated in all the normal and lymphoblastoid cells analyzed (peripheral blood mononuclear cells, Molt 4, CEM, 8402, Namalwa), after stimulation with tumor promoter agents (phorbol 12-myristate 13-acetate, phorbol 12,13-dibutyrate, mezerein). This phosphorylation is rapid (within 1-5 min), dose-dependent and results in the incorporation of PO(3-)4 groups on serine residues. Furthermore, the level of CDw50 phosphorylation induced by tumor promoter agents is decreased by the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. Activation of peripheral lymphocytes with concanavalin A, phytohemagglutinin and cross-linking of CD3 molecules also induces CDw50 phosphorylation, but the response is delayed and less intense than when tumor promoting agents are used. Treatment with any of the aforementioned agents is not accompanied by quantitative changes in the CDw50 surface expression. We therefore conclude that protein-kinase-C-mediated mechanisms are involved in phosphorylation, but not in regulation of the surface expression of the CDw50 leukocyte antigen.