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PubMed1996

Human immune response to pneumococcal polysaccharides: complement-mediated localization preferentially on CD21-positive splenic marginal zone B cells and follicular dendritic cells.

A functionally intact spleen with a marginal zone, containing B cells with high density of surface C3d-receptors (CD21), is essential for the ability to induce a primary immune response to thymus-independent type 2 (TI-2) antigens. Main representatives of natural TI-2 antigens are capsular pneumococcal polysaccharides (PPSs). In this study the localization of different types of PPS antigen is determined in human spleen tissue. Our findings indicate that a main type of TI-2 antigen, PPS, localizes preferentially in the marginal zone. PPSs show co-localization with C3, presumably C3d, at the surface of strongly CD21+ B cells equipped for rapid activation. This enables a rapid primary humoral response. The other main PPS localization at follicular dendritic cells in germinal centers, relevant for isotype switching of anti-PPS antibodies, does not seem to be dependent on the presence of specific immunoglobulin. This may explain the finding of specific IgG in an early stage after antigenic challenge. It seems likely that complement C3 fragments (likely C3d), bound to PPSs, enable PPS localization at B-cell and follicular dendritic cell surfaces by binding to CD21, the C3d receptor.

Adolescent↗
PubMedDate not supplied

C-type lectins and galectins mediate innate and adaptive immune functions: their roles in the complement activation pathway.

In recent years, a 'new' pathway for complement activation mediated by the mannose-binding lectin (MBL) has been described as a key mechanism for the mammalian acute phase response to infection. This complement activation pathway is initiated by a non-self recognition step: the binding of a humoral C-type lectin [mannose-binding lectin (MBL)] to microbial surfaces bearing 'foreign' carbohydrate determinants. The recognition factor, MBL, is associated with a serine protease [MBL-associated serine protease (MASP)] which, upon MBL binding to the microbial ligand, activates the complement component C3, leading to either (a) phagocytosis of the opsonized target via the complement receptor, or (b) humoral cell killing via assembly of the membrane attack complex. Galectins (formerly known as S-type lectins) modulate activity of the complement receptor 3 (CR3), the macrophage membrane receptor for complement components C3b and iC3b, downstream products of the MBL pathway which are covalently bound to 'target cells. Galectins also mediate macrophage- and dendrocyte-adhesion to lymphocytes activated by signaling through another C-type lectin, the L-selectin, leading to immunoglobulin-mediated responses. Thus, the functional interplay of MBL, galectins and L-selectin in the acute phase response neutralizes the microbial challenge, and lead to further adaptive immunity. Although the observation of various components of the lectin pathway in different invertebrate species demonstrates the high conservation and ancient roots of the components of innate immunity, there has previously been no evidence supporting the possibility that the integral lectin-mediated complement activation pathway is present in invertebrates. We now have evidence for the coexistence of homologs of all the pathway's key components (MBL, MASP, C3, and galectin) in the protochordate Clavelina picta, suggesting the lectin-mediated pathway of complement activation preceded the immunoglobulin pathway in evolution. Therefore, despite being 'new' to the textbooks, experimental evidence indicates that this pathway is ancient, and has been conserved intact throughout its evolution.

Amino Acid Sequence↗
PubMed1997

Analysis of the human CD21 transcription unit reveals differential splicing of exon 11 in mature transcripts and excludes alternative splicing as the mechanism causing solubilization of CD21.

CD21 is found in a soluble form at low levels in normal human sera and at elevated levels in sera from patients with EBV-associated diseases and B-CLL. Ablation of complement, injection of recombinant soluble CD21 and knock-out of CD21 in mice by gene targeting interfere with T-cell-dependent immune responses, suggesting that in vivo-generated soluble CD21 may exert immunoregulatory functions. Soluble CD21 has a molecular weight of 130,000/135,000, which is equivalent to the entire extracellular domain. Soluble forms of membrane-anchored molecules may be generated by proteolytic cleavage of the extracellular portion or by the exclusion of the hydrophobic transmembrane region via alternative splicing. To delineate whether alternative splicing of CD21 mRNA creates transcripts encoding for the soluble form of CD21 we analyzed by PCR CD21 expression in PBLs, spleen, tonsils, bone marrow and in various cell lines. We found that all CD21 mRNA species contained the transmembrane exons, thus excluding alternative splicing as a factor contributing to the serum pool of soluble CD21. Differential splicing of the CD21 transcription unit has also been suggested for exon 11. Within the CD21 gene exons 3, 7 and 11 have a high degree of homology. However, we found in malignant human cell lines and primary lymphocytes from blood, bone marrow, tonsils and spleen that exon 11, but not exon 3 or 7, is subject to alternative splicing. We cloned and sequenced the intron preceding exon 11 and found that the surrounding splice sites match consensus splice sites. In conclusion, we show that human CD21 exon 11 is alternatively spliced in malignant cell lines of lymphoid origin and in purified lymphocytes from blood, tonsils, bone marrow and spleen. We found that both exon 11 lacking and exon 11 containing transcripts are always coexpressed and the ratio of the two forms is > 1. Moreover, we exclude the possibility that alternative splicing of the transmembrane region is the mechanism leading to soluble CD21.

Alternative Splicing↗
PubMed1998

Evidence for a new transcript of the Epstein-Barr virus/C3d receptor (CR2, CD21) which is due to alternative exon usage.

CR2 extracellular domain is constituted of 15 or 16 Short Consensus Repeats (SCR), with additional SCR 11 localized between SCRs 10 and 12. We amplified Raji cDNA library, with specific primers where SCR 11 is localized. This generated a new fragment of 643 bp (16b SCR), in addition to the two expected transcripts of 489 (15 SCR) and 667 (16a SCR) bp. Sequencing these three fragments and the corresponding genomic DNA, demonstrated the presence of a 24 bp deletion in 16b SCR, without change of open reading frame and that this 24 bp region was flanked by two splicing acceptor sites. This supported a new alternative splicing of CR2, with generation of a third distinct mRNA. This third transcript was expressed in human CR2 positive T cells, normal or transformed B cells and EBV negative B cell lines. The 24 bp deletion corresponds to a proline-rich region, which may influence CR2 conformation and more likely have consequences on CR2 extra and intracellular interactions.

Alternative Splicing↗
PubMed1999

Functional characterization of soluble and membrane-bound forms of vaccinia virus complement control protein (VCP).

Vaccinia virus secretes a 35 kD protein, vaccinia virus complement control protein (VCP), that inhibits the classical and alternative pathways of complement at several points, indicating that it may be a viral analogue of human complement receptor type 1 (CR1; CD35). Structurally, however, CR1 is composed of 30 short consensus repeats (SCRs), whereas VCP consists entirely of four SCRs. We have begun a structure-function analysis of VCP to define the minimum number of SCRs necessary for function, the functional differences between VCP and CR1, and the potential therapeutic roles for VCP. We addressed these questions by creating and characterizing recombinant soluble and membrane-bound forms of VCP. We have determined that (1) VCP requires all four SCRs to bind C3b, (2) whereas CR1 binds C3b and iC3b, VCP binds C3b but not iC3b, and (3) although normally secreted, if expressed on the membrane of mammalian cells, VCP effectively protects the cells from complement-mediated lysis. Thus, VCP appears to be a compact and unique complement regulatory protein with the ability to inhibit both arms of the complement cascade, but lacking affinity for iC3b. By releasing rather than capturing iC3b-bearing complexes following inactivation of C3b, VCP may 'recycle' its active site locally among infected cells, and thereby enable the virus to evade more efficiently host immune and inflammatory responses. The unique function, compact structure, and capacity of VCP to protect mammalian cells from complement-mediated attack, suggests that it could be used both to better understand the structure-function relationship of complement regulatory proteins, in general, and also to rationally design and develop novel therapeutic agents.

Animals↗
PubMed2000

Immunomodulatory functions of murine CR1/2.

C3-fragments generated upon complement activation play an important role in the formation and regulation of immune responses. Receptors interacting with various activation fragments of this versatile complement component are expressed on a wide variety of cell types, such as lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes, consequently C3-products may influence several biological functions at different sites of the body, where complement activation takes place. In the last decade, genes, protein structure and functions played by murine complement receptors CR1 and CR2 (mCR1/2) have been deciphered. In this review, we wish to relate these properties, and fit it into the context of events following in vivo complement activation. We separately address the roles played by murine mCR1/2 as BCR coreceptor and as BCR independent structure, and propose a mchanism for the utilization of antigen-C3d conjugates bound on B cells. Finally, we raise some of the questions that remain to be elucidated in order to get a more precise picture of the functions of mCR1/2.

Animals↗
PubMed1997

Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector.

We have made use of the plasmid vector pBSV-8His recently established for baculovirus-mediated expression of His-tagged proteins for the production of a truncated soluble complement regulator protein. The protein comprised the N-terminal part, i.e. short consensus repeats (SCRs) 1-4, of the B-cell membrane protein complement receptor type two (CR2; CD21) and contained the functional epitopes which mediate the binding of the complement component C3 fragments C3dg and iC3b. This recombinant protein, termed rsCR2.1-4, was furnished with a C-terminal histidine-tag for easy purification from insect cell supernatant. The yield of > 90% pure rsCR2.1-4 was 3 micrograms/ml supernatant at day eight p.i. RsCR2.1-4 was expressed as two proteins with a M(r) of 29 and 31 representing differentially glycosylated forms. Both reacted specifically with anti-CR2 mAb HB5 directed against SCRs 3-4, but not with anti-CR2 mAbs recognizing SCRs beyond SCR 4. RsCR2.1-4 was able to bind C3dg and to block binding of C3dg-coated beads to Raji cells. Used as antigen for immunization, it allowed the efficient and well-aimed generation of antisera which specifically blocked attachment of C3dg-coated beads to Raji B cells. Thus, insect cell derived rsCR2.1-4 has proved a valuable tool to study the functional domain of CR2 and its immunoregulatory capacity in B-lymphocytes.

Animals↗
PubMed1999

Targeting of influenza epitopes to murine CR1/CR2 using single-chain antibodies.

Single-chain variable fragment (scFv) antibodies are genetically engineered molecules comprising the variable regions responsible for specific binding. scFv that recognize certain surface molecules on professional antigen presenting cells could therefore be suitable for targeting Ag to these cells. We have produced an scFv that recognizes murine complement receptors 1 and 2 (CR1/CR2) and genetically fused it with different numbers of influenza hemagglutinin peptides which contain both B and T cell epitopes. The CR1/CR2 specific hybridoma 7G6 was used for RT-PCR to obtain the variable regions, which were then combined to create an scFv fragment. The influenza hemagglutinin intersubunit peptide HA317-41 (IP) was engineered to the N terminus of the scFv in one, two or three copies. The so obtained IP(1-3)7G6scFv still bound the complement receptors; the peptides in the construct were recognized by the peptide specific monoclonal IP2-11-1 on Western blots and ELISAs. The CR1/CR2 positive B lymphomas A20 and 2PK3 presented the peptide to an I-Ed restricted IP specific T cell hybridoma more efficiently when incubated with the IP(1)7G6 constructs as compared to the free peptide. The results suggest that scFv could work as targeting devices in subunit vaccines.

Amino Acid Sequence↗
PubMed1999

Functional properties of soluble CD21.

Soluble receptors may display immunoregulatory properties by blocking interactions between ligands and their membrane receptors or by triggering specific biologic responses through interaction with counter part membrane receptors. A natural soluble form of CD21 that is cleaved from lymphocyte membrane CD21 circulates in normal human serum. Soluble CD21 retains the capacity to bind iC3b and CD23, the known ligands of membrane CD21. In a similar fashion to IgE complexes, another ligand of CD23, the soluble CD21 was shown to efficiently trigger CD23-signalling pathways in human monocytes. By inducing release of proinflammatory cytokines and upregulating expression of molecules involved in antigen presentation, soluble CD21 modulates critical monocyte functions that may be relevant to allergic and inflammatory disorders.

Humans↗
PubMed2001

Lymphocyte subsets in peripheral blood of dogs--a flow cytometric study.

Slight differences in the results of papers describing lymphocyte subsets distribution in the peripheral blood of healthy dogs may be explained by differences in monoclonal antibody clones and sources, breed and age of animals examined, methods of sample treatment, or methods of result analysis. In this paper, we described the effect of sample processing and of sample storage as well as the effect of age, breed, and gender of dogs on lymphocyte subset distribution. No significant differences were found between samples processed following a whole-blood lysis method and samples processed after density gradient separation. Furthermore, no significant differences were found between samples processed within 2h after collection and those stored at 4 degrees C for 12-16 h before processing. Age-related changes were evident in lymphocyte subset distribution in the peripheral blood of 38 Beagles divided according to their age into the six groups: (1) 5-6 days; (2) 2 months; (3) 6 months; (4) 1-2 years; (5) 3-5 years; and (6) >5 years. The percentage of B-lymphocytes (CD21-like positive cells) in the peripheral blood of newborn pups was 39.5+/-5.7 and decreased with advancing age. The percentage of CD8+ lymphocytes was 7.7+/-3.4 after birth and increased with advancing age. No age-related changes were observed in the percentages of CD4+ lymphocytes. The CD4+:CD8+ ratio decreased with advancing age. No significant age-related change was observed for lymphocytes bearing the gammadelta-TCR. Some breed differences were evident. Adult (1-5-year-old) Beagles, German Shepherds, Dalmatians, and Dachshunds were examined. The percentages of lymphocytes were higher in Beagles and Dachshunds than in Dalmatians and German Shepherds. The highest and the lowest absolute lymphocyte counts were found in Beagles and German Shepherds, respectively. As a consequence, German Shepherds showed the lowest absolute counts of the individual lymphocyte subpopulations and the widest neutrophil:lymphocyte ratio. Dalmatians showed the lowest percentage of CD3+ cells, the highest percentage of CD21+ cells, and the lowest CD4+:CD8+ ratio. German Shepherds showed the lowest percentage of CD21+ cells and the highest CD4+:CD8+ ratio. Females in Beagles and Dachshuns had nonsignificantly higher percentages of total lymphocytes, CD3+, CD4+, and nonsignificantly lower percentages of CD21+ lymphocytes. We concluded that there are age-, breed-, and perhaps also gender-related differences in lymphocyte subset distribution in the peripheral blood of dogs. Therefore, there is need to use appropriate control group in the experimental protocols. Among-breed differences could explain, at least partly, breed predisposition for some diseases.

Age Factors↗
PubMed2002

Monoclonal antibodies to lymphocyte surface antigens for cetacean homologues to CD2, CD19 and CD21.

CD2 is a pan-T cell marker, while CD19 and CD21 are important molecules in signal transduction of B lymphocytes. CD19 and CD21 are both present on mature B cells, while CD19 is also present in developing B cells and plasma cells. Monoclonal antibodies (mAbs) against cetacean lymphocyte putative homologues to CD2 (two different antibodies), CD19 and CD21 were characterized. The proteins immunoprecipitated were as follows: F21.I (putative anti-CD2), 43 and 59kDa; F21.B (putative anti-CD19), 83 and 127kDa; F21.F (putative anti-CD21), 144kDa. The second putative anti-CD2 (F21.C) selectively inhibited the binding of F21.I. Both the putative anti-CD2 (T cell markers) stained T-cell zones on lymph node sections, while both the B cell markers (putative CD19 and CD21) stained B-cell zones. F21.B and F21.F were absent from thymus single cell suspension but labeled 63 and 65% mesenteric lymph node lymphocytes, respectively, while both F21.C and F21.F were present on 100% thymocytes and fewer lymph node lymphocytes. B and T cell markers were mutually exclusive on double labeling using flow cytometry. These mAbs are foreseen as possible valuable diagnostic and research tools to assess immune functions of captive and wild cetaceans.

Animals↗
PubMed2002

IgE is present on peripheral blood monocytes and B cells in normal dogs and dogs with atopic dermatitis but there is no correlation with serum IgE concentrations.

Blood was collected from 29 dogs, 14 with atopic dermatitis (AD) and 15 controls. Total serum IgE was quantitated. Peripheral blood monocytes were harvested and labeled with leucocyte markers and anti-canine IgE before analysis by flow cytometry. There was no statistically significant difference between the atopic and control groups when the mean number of cells in the monocyte (CD14), antigen presenting cell (CD1c) or B cell (CD21) populations were examined. However, the variation in cell numbers was significant and much greater in the atopic group for CD1c and CD14 labeled cells. The mean percentage of double labeled cells, CD1c/IgE and CD14/IgE was significantly lower in the atopic population compared with the controls. More variation was observed in the numbers of monocytes of atopic dogs (CD14/IgE) and antigen presenting cells (CD1c/IgE) of control dogs. The mean percentage of B cells expressing IgE was 65 and 51% in the atopic and control groups respectively which is greater than that reported in humans. There was no statistically significant difference. Total serum IgE concentrations were similar in each group and did not correlate with cell bound IgE in any of the leucocyte populations studied. Canine AD is associated with more variability in circulating monocyte numbers and lower numbers of monocytes expressing IgE than control dogs. Unlike in humans, there is no correlation between circulating and cell bound IgE. Furthermore, high levels of IgE in the dog may be related to a greater number of B cells in the circulation committed to IgE production.

Animals↗
PubMed2002

Up-regulation by feline interleukin-4 and down-regulation by feline interferon-gamma of major histocompatibility complex class II on cat B-lymphocytes.

Interleukin-4 (IL-4) exhibits numerous biological and immunoregulatory functions on B- and T-lymphocytes, monocytes, and dendritic cells in both mice and humans. In the present study, we show that IL-4 also has a regulatory function in the cat species. Cells transfected with IL-4 DNA expressed a biologically active protein as demonstrated by the up-regulation of MHC class II molecules on B-lymphocytes (CD21(+)) in a flow cytometric assay. Increased levels of MHC class II expression on CD21(+) cells were seen in 11 out of 12 cats (p<0.05). In addition, 12 out of 12 cats showed up-regulation of MHC class II on CD21(-) cells, mainly consisting of T-lymphocytes (p<0.05). In contrast, concanavalin A (ConA)-induced culture supernatant from peripheral blood mononuclear cells (PBMCs) containing high levels of interferon-gamma (IFN-gamma) transcripts induced down-regulation of MHC class II molecules on CD21(+) cells of all samples (p<0.05). Variable results were observed for CD21(-) cells incubated with ConA-conditioned medium (p=0.71). The nature of the cytokine(s) responsible for these effects remains to be determined. However, the fact that down-regulation of MHC class II molecules on B cells occurred in all cats tested suggests that IFN-gamma may be involved. These data provide further insight into the mechanism by which MHC class II expression is regulated in feline lymphocytes, and suggest that the Th1/Th2 paradigm is also present in the cat.

Animals↗
PubMed2003

Comparison of flow cytometry and image morphometry in the quantitative analysis of cell population markers in the lymph node of sheep.

Two approaches to the quantitative analysis of cell population markers in tissues are flow cytometry and image morphometry. To compare these methods, sheep lymph nodes were collected and analysed for CD8+ and CD21+ cell populations, which were selected to represent dispersed and concentrated cell populations, respectively. These two populations were measured as a percentage of total cell count (flow) or total tissue area (morphometry). The two populations were also measured as a percentage of respective base populations (CD2+ cells for CD8 and MHC II+ cells for CD21). A simple linear regression analysis showed that when the cell populations were assessed as a percentage of total cell count or total area, measurements obtained with flow and morphometry only correlated significantly with the dispersed CD8+ population and not with the highly concentrated CD21+ population. However, when the cell populations were assessed as a percentage of their base population, measurements obtained with flow and morphometry showed a significant correlation for both the dispersed and concentrated cell populations. This study demonstrates that measurements of lymph node cell populations obtained with the two methods are comparable, but that tissue distribution of cell populations should be considered, when the unit of measurement is chosen.

Animals↗
PubMed1999

Structure and expression of ovine complement receptor type 2.

The structure of sheep complement receptor type 2 (CR2) was characterised by cDNA cloning, protein sequencing and immunoprecipitation. The primary structure of sheep CR2 is similar to known mammalian homologues but the higher-order structure is unusual. Two distinct CR2 isoforms occur, one of which is ubiquitinated in the cytoplasmic domain, and the two molecular forms are expressed at the cell surface as non-covalently associated dimers. The percentage of sheep B-cells that express CR2 changes during development and varies between different body compartments. CR2+ and CR2 B-cell subsets also differ in the expression of other surface markers and in functional properties. Differential expression of CR2 may, therefore, delineate B-cells that arose by alternative developmental pathways, or it could be a marker for B-cells at different phases of antigen exposure.

Animals↗
PubMed2002

Mucosal type mast cells express complement receptor type 2 (CD21).

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.

Animals↗
PubMed2002

Role for complement receptors (CD21/CD35) in the regulation of recombination activating gene expression in murine peripheral B cells.

A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect on immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells.

Animals↗
PubMed1992

Properties of soluble CR2 in human serum.

A soluble form of complement receptor number 2 (sCR2) found in human serum closely resembles that produced in culture by B lymphoblastoid cells. Epitope analysis with a panel of CD21 monoclonal antibodies revealed only minor differences between antigen from the two sources. Purified sCR2 from both sources bound to C3dg prepared from human or mouse serum and to u.v.-inactivated Epstein-Barr virus. SDS-PAGE analysis of culture supernates of B-lymphoid cells labelled by growth in medium containing 35S-methionine revealed a major component of molecular weight approximately 130 kDa and another band at 30 kDa. Incubation with endoglycosidase F reduced the size of the high molecular weight component. Gel filtration of untreated serum or culture supernate revealed that, in its native state, sCR2 behaved as a molecule or complex of apparent molecular weight 320 kDa. Possible explanations are discussed.

B-Lymphocytes↗