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D T Fearon

Publications and source records attributed to D T Fearon.

At least 19 recordsLinked to original sources
PubMed1992

CD19: lowering the threshold for antigen receptor stimulation of B lymphocytes.

Lymphocytes must proliferate and differentiate in response to low concentrations of a vast array of antigens. The requirements of broad specificity and sensitivity conflict because the former is met by low-affinity antigen receptors, which precludes achieving the latter with high-affinity receptors. Coligation of the membrane protein CD19 with the antigen receptor of B lymphocytes decreased the threshold for antigen receptor-dependent stimulation by two orders of magnitude. B lymphocytes proliferated when approximately 100 antigen receptors per cell, 0.03 percent of the total, were coligated with CD19. The B cell resolves its dilemma by having an accessory protein that enables activation when few antigen receptors are occupied.

Animals↗
PubMed1991

Determination of the structural basis for selective binding of Epstein-Barr virus to human complement receptor type 2.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that selectively infects and immortalizes human B lymphocytes. One determinant of this narrow tropism is human CR2, the only viral receptor within the superfamily of proteins that contain short consensus repeats (SCRs). Human CR2 serves as a receptor for both C3dg and the gp350/220 glycoprotein of EBV, and binds the monoclonal antibody (mAb) OKB7, which blocks binding of both ligands to the receptor. In contrast, although murine CR2 is capable of binding human C3dg and this interaction can be blocked with the mAb 7G6, it does not bind OKB7 or EBV. We have determined the structural basis for absolute specificity of EBV for human CR2 through characterization of a panel of 24 human-murine chimeric receptors, all of which bind human C3dg. The results indicate that preferential binding of EBV to human CR2 is not due to unique amino acids that are capable of binding the virus, but reflects a distinct receptor conformation that can be achieved in murine CR2 with single amino acid substitutions in two discontinuous regions of the primary structure: replacement of proline at position 15 with the corresponding serine from human CR2, and elimination of a potential N-linked glycosylation site between SCR-1 and SCR-2. Furthermore, species-specific binding of EBV, OKB7, and 7G6 can all be manipulated through substitutions among residues 8-15, suggesting that this octapeptide is part of a structural determinant that is critical for binding of both viral and natural ligands to CR2.

Amino Acid Sequence↗
PubMed1991

Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor.

CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.

Animals↗
PubMed1991

The CD19 complex of B lymphocytes. Activation of phospholipase C by a protein tyrosine kinase-dependent pathway that can be enhanced by the membrane IgM complex.

We have investigated the mechanism by which the membrane protein complex of the B lymphocyte that contains CD19 and CR2 activates phospholipase C (PLC) to induce a rise in [CA2+]i. The CD19 complex resembled the membrane IgM complex in that three protein tyrosine kinase inhibitors suppressed increases in [Ca2+]i and inositol bisphosphate and inositol triphosphate generation. However, the activation of PLC by the CD19 complex could be distinguished from that by the membrane IgM complex by slower kinetics of generation of inositol phosphates, resistance to inhibition by activators of protein kinase C, and different pattern of tyrosine-phosphorylated cellular substrates. Western blot analysis of lysates from cells stimulated by the CD19 complex demonstrated a single new phosphotyrosine-containing protein of 85 kDa, whereas multiple other phosphotyrosine-containing proteins were present in cells activated by the mIgM complex. In particular, PLC-gamma 1, which is a substrate for the protein tyrosine kinase activated by the mIgM complex, was not tyrosine-phosphorylated in cells stimulated by the CD19 complex. Cross-linking the two complexes together caused a synergistic increase in [CA2+]i which was neither suppressed by activation of protein kinase C nor associated with increased tyrosine-phosphorylation of PLC, characteristic of the CD19 pathway. Therefore, the B cell has two signal transduction complexes, associated with membrane IgM and CD19, that activate PLC by different mechanisms and that can synergistically interact to enhance this function by the CD19 pathway.

Antigens, CD↗
PubMed1991

Suppression of the immune response by a soluble complement receptor of B lymphocytes.

The CD19-CR2 complex of B lymphocytes contains proteins that participate in two host-defense systems, the immune and complement systems. The ligand for the subunit of the immune system, CD19, is not known, but the complement receptor subunit, CR2 (CD21), binds activation fragments of the C3 component of the complement system and may mediate immunopotentiating effects of complement. A recombinant, soluble CR2 was prepared by fusing the C3-binding region of the receptor to immunoglobulin G1 (IgG1). The (CR2)2-IgG1 chimera competed with cellular CR2 for C3 binding and suppressed the antibody response to a T cell-dependent antigen when administered to mice at the time of immunization. This inhibitory effect of (CR2)2-IgG1 demonstrates the B cell-activating function of the CD19-CR2 complex and suggests a new method for humoral immunosuppression.

Amino Acid Sequence↗
PubMed1991

Interaction of iC3b with recombinant isotypic and chimeric forms of CR2.

CR2 is a component of a signal transduction complex on B lymphocytes that augments B cell responses to Ag. We have quantitatively assessed binding by the two isotypic forms of CR2 for two of its ligands, the polymerized iC3b (p(iC3b)) fragment of C3, and gp350/220, the EBV membrane protein. The recombinant 15-SCR or 16-SCR forms of CR2 bound p(iC3b) with identical affinities. Full binding activity of CR2 for p(iC3b) was observed with a chimera comprised of SCR-1 and -2 of CR2 fused to SCR-17 through -30 of CR1. Therefore, the alternatively spliced SCR-10a has no role in binding p(iC3b), and the binding activity of wild type receptor for iC3b can be reconstituted with SCR-1 and -2 of CR2. The binding affinities of the two isoforms of CR2 for soluble gp350/220 were also similar. Additional sites in the C3c region of C3 have been postulated also to interact with CR2. However, monomeric iC3b and C3d were equally effective in inhibiting the binding of p(iC3b) to CR2, indicating that the C3c region of iC3b does not contribute to the interaction of iC3b with CR2. Finally, the relative abilities of C3b and iC3b to bind to CR1 and CR2 were compared. The conversion of C3b to iC3b generated a ligand with an approximate 100-fold decrease in affinity for CR1 and a 10-fold increased affinity for CR2, resulting in a 1000-fold greater likelihood for binding to the latter receptor that may then promote B cell activation.

Animals↗
PubMed1991

Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex.

The complement system augments the humoral immune response to low concentrations of antigen. This effect may be partly mediated by complement receptors on the surface of B lymphocytes that bind immunogenic complexes bearing fragments of C3 and C4. We have shown by immunoprecipitation analysis that the two complement receptors expressed by B lymphocytes, complement receptor 1 (CR1) and CR2, form a detergent-sensitive complex on the surface of tonsillar B lymphocytes and on K562 erythroleukemia cells that were co-transfected with cDNAs encoding CR1 and CR2. The CR1/CR2 complex is distinct from the CR2/CD19 complex and may assist B cell activation by efficiently capturing C3b-containing immunogens and maintaining such immunogens on the B cell after CR1 and factor I-mediated cleavage to iC3b and C3dg. The complement activating immunogen may then trigger signal transduction by the CR1/CR2 complex, the CR2/CD19 complex, or membrane immunoglobulin.

Antigens, CD19↗
PubMed1991

Tyrosine phosphorylation of phospholipase C induced by membrane immunoglobulin in B lymphocytes.

Ligation of membrane IgM on B lymphocytes causes activation of a protein-tyrosine kinase(s) (PTK) and of phospholipase C (PLC). To determine whether these are elements of a common signal-transduction pathway, the effect of three PTK inhibitors on the rise in intracellular free Ca2+ concentration [( Ca2+]i) in human B-lymphoblastoid cell lines was assessed. Tyrphostin completely suppressed the increase in [Ca2+]i and the generation of inositol phosphates induced by ligation of membrane immunoglobulin (mIg) M. Herbimycin and genistein reduced by 30% and 50%, respectively, the rise in [Ca2+]i caused by optimal ligation of mIgM, and they abolished it in cells activated by suboptimal ligation of mIgM. Tyrphostin had no effect on the capacity of aluminum fluoride to increase [Ca2+]i. To determine whether a function of PTK is the phosphorylation of PLC, immunoprecipitates obtained with anti-phosphotyrosine from detergent lysates of B-lymphoblastoid cells were assayed for PLC activity. Ligation of mIgM increased immunoprecipitable PLC activity 2-fold by 90 sec and 4-fold by 30 min. Specific immunoprecipitation and Western blot analysis identified tyrosine phosphorylation of the gamma 1 isoform of PLC after 60 sec of stimulation. Activation of PLC in B cells by mIgM requires PTK function and is associated with tyrosine phosphorylation of PLC-gamma 1, suggesting a mechanism of PLC activation similar to that described for certain receptor PTKs.

Aluminum↗
PubMed1991

Intersection of the complement and immune systems: a signal transduction complex of the B lymphocyte-containing complement receptor type 2 and CD19.

The complement system augments the humoral immune response, possibly by a mechanism that involves the B lymphocyte membrane receptor, CR2, which binds the C3dg fragment of C3 and triggers several B cell responses in vitro. The present study demonstrates that CR2 associates with a complex of membrane proteins that may mediate signal transduction by ligated CR2. Monoclonal antibodies to CR2 immunoprecipitated from digitonin lysates of Raji B lymphoblastoid cells a membrane complex containing CR2, approximately equimolar amounts of CD19, which is a member of the immunoglobulin superfamily, and three unidentified components: p130, p50, and p20. The complex, which was immunoprecipitated also with anti-CD19, could be dissociated by Nonidet P-40, accounting for its absence in previous studies of CR2. Expression of recombinant CR2 and CD19 in K562 erythroleukemia cells led to formation of a complex that contained not only these two proteins but also p130, p50, and p20, and another component, p14. These unidentified components of the CR2/CD19 complex coimmunoprecipitated with CD19 and not with CR2 from singly transfected cells, indicating primary association with the former. CD19 replicated the capacity of CR2 to interact synergistically with mIgM for increasing free intracellular Ca2+, suggesting that the complex mediates this function of CR2. Therefore, CR2 associates directly with CD19 to become a ligand-binding subunit of a pre-existing signal transduction complex of the B cell that may be representative of a family of membrane protein complexes. This interaction between the complement and immune systems differs from that between immunoglobulin and Clq by involving membrane rather than plasma proteins, and by having complement involved in the afferent phase of the immune response.

Antigens, CD↗
PubMed1991

Anti-inflammatory and immunosuppressive effects of recombinant soluble complement receptors.

CR1 and CR2 have served as unusual probes for the analysis of the two major functions of the immune system involving inflammation and the immune response, respectively. CR1, or some construct containing its active site SCRs, may find a role in the therapy of complement-dependent tissue injury, and may be used to define which diseases are caused by the inappropriate or excessive activation of this system. Although soluble forms of CR2 may be shown to have potential clinical utility when foreign antigen is given prospectively, as in monoclonal antibody therapy, perhaps the most important finding emanating from the analysis of this receptor is the recognition of a previously unrecognized membrane protein complex whose role in B cell development is yet to be determined. It is reasonable to predict that the function of the CD19 complex will be significant as it serves as the link between two evolutionarily distinct systems that share a common purpose of anti-microbial host defense.

Animals↗
PubMed1990

Soluble human complement receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis.

The complement system is an important mediator of the acute inflammatory response, and an effective inhibitor would suppress tissue damage in many autoimmune and inflammatory diseases. Such an inhibitor might be found among the endogenous regulatory proteins of complement that block the enzymes that activate C3 and C5. Of these proteins, complement receptor type 1 (CR1; CD35) has the most inhibitory potential, but its restriction to a few cell types limits its function in vivo. This limitation was overcome by the recombinant, soluble human CR1, sCR1, which lacks the transmembrane and cytoplasmic domains. The sCR1 bivalently bound dimeric forms of its ligands, C3b and methylamine-treated C4 (C4-ma), and promoted their inactivation by factor I. In nanomolar concentrations, sCR1 blocked complement activation in human serum by the two pathways. The sCR1 had complement inhibitory and anti-inflammatory activities in a rat model of reperfusion injury of ischemic myocardium, reducing myocardial infarction size by 44 percent. These findings identify sCR1 as a potential agent for the suppression of complement-dependent tissue injury in autoimmune and inflammatory diseases.

Animals↗
PubMed1990

Recombinant soluble CR1 suppressed complement activation, inflammation, and necrosis associated with reperfusion of ischemic myocardium.

In summary, conversion of wild-type CR1 to a soluble form (sCR1) creates a potent inhibitor of complement activation by both the classical and alternative pathways by inhibiting the C3/C5 convertases. In the rat reperfusion infarct model, sCR1 significantly suppresses complement activation at the endothelial surface of capillaries and venules. This suppression of complement activation is accompanied by reduced accumulation of leukocytes within the infarct zone, perhaps because of reduction of the generation of C5a, which promotes expression of leukocyte adhesion receptors and leukocyte chemotaxis. In addition, formation of the C5b-9 attack complex, which may contribute to direct endothelial injury, was suppressed by sCR1. The inhibition of complement activation and leukocyte infiltration by sCR1 explains the observed significant reduction in myocardial necrosis after ischemia and reperfusion. These studies have identified sCR1 as a potential agent for therapeutic intervention in diseases associated with complement-dependent tissue injury.

Amino Acid Sequence↗
PubMed1990

Deficiencies of human C3 complement receptors type 1 (CR1, CD35) and type 2 (CR2, CD21).

CR1 (CD35) and CR2 (CD21) are structurally related integral transmembrane glycoproteins that function as cellular receptors for human C3b and C3dg, respectively. The primary sequence of the most common structural allotype of CR1 and that of CR2 have been established, and ligand binding on the molecules has been mapped. CR1 and CR2 genes are located in close vicinity in the RCA locus of chromosome 1. CR1 has a wide cellular/tissular distribution and mediates a variety of biologic functions, including the transport of C3-bearing immune complexes on erythrocytes, enhancement of phagocytosis, induction of IL-1 secretion and enhancement of B-cell differentiation. Expression of CR2 is restricted to B lymphocytes and follicular dendritic cells. The receptor modulates B-cell growth. CR2 also serves as the receptor for EBV and determines the cellular tropism of the virus. This review discusses the molecular biology and functional characteristics of CR1 and CR2. It focuses on alterations of expression of the receptors in disease, with particular emphasis on the genetic and acquired factors that contribute to the defective expression of CR1 in patients with systemic lupus erythematosus.

Amino Acid Sequence↗
PubMed1989

Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2.

Complement receptor type 2 (CR2;CD21), a member of the superfamily of proteins containing short consensus repeats (SCRs), is the B cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV), and for the C3dg protein of complement. By analysis of CR2 deletion mutants and chimeras formed with CR1 (CD35) we determined that of the 15 SCRs in CR2, the NH2-terminal two SCRs are necessary and sufficient to bind both gp350/220 and C3dg with affinities equivalent to those of the wild-type receptor. The epitope for OKB-7, a mAb that blocks binding of both EBV and C3dg and shares with these ligands B cell-activating capabilities, also requires both SCR-1 and SCR-2, whereas mAbs lacking these functions bind to other SCRs. Thus, EBV, a polyclonal activator of B cells, has selected a site that is proximate or identical to the natural ligand binding site in CR2, perhaps reflecting the relative immutability of that site as well as its signal transducing function.

Animals↗
PubMed1989

Polymeric C3dg primes human B lymphocytes for proliferation induced by anti-IgM.

Polymeric C3dg (pC3dg), having an average m.w. of approximately 400,000 and saturating complement receptor type 2 (CR2) on B lymphoblastoid cells at 1 micrograms/ml, was preincubated with tonsillar B cells for 24 h, after which anti-IgM was added and proliferation assessed by thymidine incorporation. Preculture of B cells with 0.01 to 1.0 micrograms/ml of polymerized C3dg (pC3dg) caused a dose-dependent enhancement of proliferation and accelerated entry into S phase after addition of anti-IgM. The continued presence of pC3dg during stimulation by anti-IgM was not required. pC3dg alone did not induce proliferation and preculture of B cells with C3dg monomer had no effect on the subsequent response to anti-IgM. The priming effect of pC3dg required at least 6 h and was greatest after 24 h of preculture. Preincubation with pC3dg did not lower the concentration of anti-IgM necessary for induction of proliferation, but did enhance proliferation at all concentrations above this threshold. Augmented proliferation occurred only in B cells of higher density in Percoll gradients, and neither T cells nor monocytes were required. Thus, independent interaction of CR2 with its natural ligand primes the B cell for subsequent stimulation through the Ag receptor, an effect that might synergize with the previously described CR2 function of lowering the threshold for B cell activation when crosslinked to membrane IgM.

Antibodies, Anti-Idiotypic↗
PubMed1989

Structure of the human CR1 gene. Molecular basis of the structural and quantitative polymorphisms and identification of a new CR1-like allele.

Structural and quantitative polymorphisms have been described in human CR1. In the former, the S allotype is larger than the F allotype by 40-50 kD, the size of a long homologous repeat (LHR). In the latter, homozygotes for a 7.4-kb Hind III fragment express fourfold more CR1 per erythrocyte than do homozygotes for the allelic 6.9-kb restriction fragment. The basis for these genomic polymorphisms has been determined by restriction mapping the entire S allele and part of the F allele. The S allele is 158 kb and contains 5 LHRs of 20-30 kb, designated -A, -B/A, -B, -C, and -D, respectively, 5' to 3'. Extensive homology was found among the LHRs in their restriction maps, exon organization, and the coding and noncoding sequences. The presence of LHR-B/A in the S allele but not in the F allele accounts for the longer transcripts and polypeptide associated with the former allotype. At least 42 exons are present in the S allele, with distinct exons for the leader sequence, the transmembrane and cytoplasmic regions and most of the SCRs comprising the extracellular portion of CR1. Consistent with the mapping of the ligand binding site to the first two SCRs in each LHR, the second SCRs in LHR-A, -B/A, -B, and -C are encoded by two exons, reflecting a specialized function for this unit. The allelic 7.4/6.9-kb Hind III fragments extend from the 3' region of LHR-C to LHR-D. The 6.9-kb restriction fragment is the result of a new Hind III site generated by a single base change in the intron between the exons encoding the second SCR of LHR-D. A second cluster of genomic clones has been identified by hybridization to CR1 probes. Although they contain regions of hybridization to the cDNA and genomic probes derived from CR1, these cannot be overlapped with the structural gene owing to their distinct restriction maps. Three genomic polymorphisms previously identified by CR1 cDNA probes map to this region. These additional clones may represent part of a duplicated allele located nearby within the CR1 locus.

Alleles↗