CR1 Expression Research Articles

Nonopsonic and Opsonic Association ofMycobacterium tuberculosiswith Resident Alveolar Macrophages Is Inefficient

AbstractThe association of Mycobacterium tuberculosis with alveolar macrophages (Mφ) in a serum-free environment is a crucial first step in the pathogenesis of this facultative intracellular pathogen. We present data demonstrating that freshly explanted alveolar Mφ do not efficiently bind M. tuberculosis in a serum-free system, although a small subpopulation of these Mφ (10–15%) can bind mycobacteria. In contrast, almost 100% of a peritoneal Mφ population bind mycobacteria under the same conditions. The poor binding of mycobacteria by alveolar Mφ does not reflect a general inability to associate with particles; binding and ingestion of latex beads and zymosan particles were comparable with that seen with peritoneal Mφ. Resident alveolar Mφ did not efficiently bind mycobacteria in the presence of serum and expressed poorly several Mφ surface receptors, including CR3. Furthermore, we demonstrate that bovine surfactant protein A does not enhance the association of M. tuberculosis with alveolar Mφ. Differentiation of alveolar Mφ in vitro resulted in increased expression of Mφ surface receptors and an increased capacity to bind mycobacteria in the presence and absence of serum. Evidence is presented that opsonic binding of M. tuberculosis by differentiated alveolar Mφ is mediated by complement and CR3, and that the poor binding by resident alveolar Mφ is due to their poor expression of CR3. The receptor mediating nonopsonic binding of M. tuberculosis to differentiated alveolar Mφ was not unequivocally identified in this study, but could also be CR3.

Expression and Function of the Type 3 Complement Receptor in Tissues of the Developing Mouse

Macrophage (Mphi) expression of the leukocyte integrins has been implicated in their adhesion and migration in the adult. Little is known, however, of the expression or function of these molecules during development. This study defines the spatial and temporal sequences of expression of the type 3 complement receptor (CR3) in the developing mouse; establishes the functional efficacy of this molecule in spreading, adhesion, and phagocytosis; and investigates its role in inflammatory and constitutive migration. Expression of CR3 on monocytes occurred early compared to Mphi-restricted glycoprotein F4/80, but expression on stellate tissue Mphi appeared later than F4/80 and was transient. Expression of CR3 on resident tissue Mphi is more widespread during development, being retained on only very specific Mphi populations in the adult. Neutrophil polymorphs expressed CR3 from day 17 of gestation onward. The anti-CR3 mAb 5C6 was used to investigate the role of CR3 in adhesion, spreading, and phagocytosis by neonatal Mphi. Neonatal macrophages were found to adhere, spread, and phagocytose by CR3-dependent mechanisms, and a CR3-independent system was implicated in the spreading of neonatal Mphi. The role of CR3 in migration during development was then investigated. 5C6 had potent effects on the early stages of the migration of myelomonocytic cells to an inflammatory stimulus in vivo. Despite efficient transplacental transfer of the Ab from pregnant mother to fetus, the process by which monocytes generate populations of resident tissue Mphi was undisrupted, indicating the existence of CR3-independent mechanisms of monocyte migration during development.

Cell Therapy by Using Genetic Engineered Glomerular Cells to Remove Immune Complex Deposited on Kidney

This study discloses a novel concept different from both the traditional artificial kidney and the lymphocyte cell therapy, to cure the chronic renal disease, particularly the disease of SLE (systemic lupus erythematosus), which is regarded as an autoimmune disease caused by the existance of excessive amount of immune complex in the patients’ serum. In this study, a full length of human tonsil CR1 cDNA was transfected into normal rat glomerular epithelial cells to enable the CR1 expression in a long term (more than 1 year). As compared with the non-CRl expressed cells, the CR1 expressed ones showed a higher binding effect to immune complex in a long period (more than 60 minutes), and the binding effect was demonstrated to be mediated by complement of serum. Anti-CR1 antibody blocking the binding effect indicates that CR1 plays an important role on immune-complex removal. Microsopic results showing that the genetic engineered cells possessed an enhancing phagocytic ability, and the curve of immune complex bound by these cells matched to the phagocytic equation, may suggest that the mechanism of the immune complex removal is due to the ligand-receptor binding and the phagocytosis of the genetic engineered cells. This mechanism may be applicable to the patients with SLE disease to remove the excess immune complex.

Cripto: roles in mammary cell growth, survival, differentiation and transformation.

Cripto-1 (Cr-1) protein, encoded by the teratocarcinoma-derived growth factor gene (TDGF-1), is highly correlated with transformation in breast cancer. Eighty-two percent of breast carcinomas express Cr-1 whereas it is undetected in normal human breast tissue. We confirmed and extended findings that Cr-1 protein is expressed during the pregnancy and lactating stages of normal murine mammary glands but is barely detectable in glands from virgin animals and is undetectable in involuted glands. Cr-1 was found to be expressed in CID 9 cells, a line of mammary epithelial cells derived from 14.5 day pregnant mice and we have used these cells to investigate the roles of this gene. Exogenous mouse Cr-1 expression from a retroviral vector caused CID 9 cells to grow at an increased rate and to increased cell densities compared to parental and control cells. CID 9 cells overexpressing Cr-1 did not differentiate efficiently. Infection of CID 9 cells with a Cr-1 antisense vector caused these cells to change in morphology, to grow slowly, to undergo apoptosis at a higher rate and to achieve lower saturation densities but the cells were still capable of differentiating. We concluded that Cr-1 is an autocrine growth factor for normal breast cells, that when over-expressed stimulates excessive cell proliferation at the expense of differentiation. In transplantation studies, Cr-1 over-expression stimulated the growth and survival of mammary cells, but did not stimulate tumorigenesis in vivo.

Role of complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) in murine lupus

A previous study has demonstrated that complement receptors on the surface of polymorphonuclear leucocytes (neutrophils) in gingival crevicular fluid significantly increased compared with those in autologous peripheral blood obtained from periodontitis-affected subjects. The present study attempted to determine the mRNA levels of complement receptor types 1 and 3 (CR1, CR3) on neutrophils in gingival crevicular fluid using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Gingival crevicular fluid samples were obtained from 11 adult periodontitis patients by gingival crevicular washing, and venipunctured peripheral blood was used as a control. RT-PCR analysis was performed using the primer sets for CR1, CR3 and β-actin. Digoxigenin-labelled RNA probes were synthesized from RT-PCR products for in situ hybridization. Both CR1 and CR3 mRNA levels relative to β-actin were significantly lower in crevicular fluid neutrophils than in peripheral blood neutrophils (crevicular fluid-CR1: 32.75±22.93%, peripheral blood-CR1: 65.30±43.25%, p < 0.005; crevicular fluid-CR3: 9.09 ± 5.34%, peripheral blood-CR3: 30.14 ± 18.80%, p < 0.005). In in situ hybridization, a greater majority of neutrophils showed positive CR1 and CR3 mRNA expression, while only a few neutrophils showed positive signals in gingival crevicular fluid. Data in the present study suggest that increased expression of complement receptors on the neutrophil cell surface appears to be unrelated to de novo synthesis.

An Intronic Silencer Regulates B Lymphocyte Cell- and Stage-Specific Expression of the Human Complement Receptor Type 2 (CR2, CD21) Gene

Human CR2 (CD21) is a B lymphocyte protein whose surface expression is restricted primarily to the mature cell stage during development. To study the transcriptional mechanisms that govern cell- and stage-restricted CR2 expression, we first performed transient transfection analysis using constructs extending from -5 kb to +75 bp (-5 kb/+75) in the CR2 promoter. The promoter was found to be broadly active, with no evidence of cell- or stage-specific reporter gene expression. However, the addition of a 2.5-kb intronic gene segment (containing a DNase I hypersensitive site) to the (-5-kb/+75) construct resulted in appropriate reporter gene expression, defined as the silencing of the (-5-kb/+75) promoter activity only in non-CR2-expressing cells. Interestingly, appropriate reporter gene expression required stable transfection of the constructs in cell lines, suggesting nuclear matrix or chromatin interactions may be important for appropriate CR2 gene expression. Importantly, transgenic mice also required the intronic silencer to generate lymphoid tissue-specific reporter gene expression. Some transgenic founder lines did not demonstrate reporter gene expression, however, indicating that additional transcriptional regulatory elements are present in other regions of the CR2 gene. In summary, these data support the hypothesis that human CR2 expression is regulated primarily by an intronic silencer with lineage- and B cell stage-specific activity.

The requirement of localized, CR2-mediated, alternative pathway activation of complement for covalent deposition of C3 fragments on normal B cells.

We have shown previously that normal B cells share, with Epstein-Barr virus-transformed and malignant B cells, the ability to activate the alternative pathway (AP) of complement in vitro, resulting in the deposition of C3 fragments on the cell surface. Complement receptor type 2 (CR2, CD21) has been implicated directly as the site for formation of an AP convertase, which provides nascent C3b for deposition at secondary sites on the B-cell surface. In the present study, we have examined C3 fragment deposition in vitro in more detail by (1) assessing the importance of locally generated C3b for the deposition process, (2) investigating whether CR2 is the sole requirement for conferring AP activation capacity on a cell, and (3) determining whether CR2's function, as an AP activator, has different structural requirements from ligand binding. Increasing the availability of native C3, by increasing the serum (NHS) concentration, resulted in enhanced C3 fragment deposition on the B cells, whereas use of factor 1-depleted NHS, which showed massive fluid phase C3 conversion during the incubation, diminished the deposition. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting of untreated and hydroxylamine-treated lysates from B cells, after in vitro activation, revealed that the majority of C3 fragments (primarily iC3b and C3dg) had been covalently bound to the cell surface. Transfection of COS cells with wild-type CR2 or a deletion mutant lacking 11 of the molecule's 15 homologous domains, but retaining the ligand-binding site, revealed that expression of intact CR2 conferred a 12-fold increase in AP-activating capacity on these cells, while no increase in AP activity was apparent on cells transfected with the mutant CR2.

Relationship between glomerular epithelial cell injury and proteinuria in IgA nephropathy

The present study was designed to elucidate whether glomerular epithelial cell (GEC) injury is associated with the mechanism of proteinuria in biopsy-proven IgA nephropathy (IgAN). Twenty-four IgAN patients were divided into 4 groups based on their grade of proteinuria. Light microscopic examination revealed that GEC injury appeared prior to glomerular lesions such as sclerosis, crescent and adhesion. Reduced glomerular expression of C3b receptor (CR1), an indicator of GEC injury, was detected initially at the portion of damaged GEC and around the lesions of sclerosis, crescent and adhesion. CR1 expression eventually disappeared in the group IV patients who had nephrotic range proteinuria. Moreover, the grade of proteinuria in IgAN patients was associated with GEC injury and reduced glomerular expression of CR1. Taken together, GEC injury might be an important pathological finding which appears initially in the glomeruli, suggesting that GEC injury is a predictor of proteinuria in IgAN patients.

Immunohistological studies on macrophages in lymph nodes of onchocerciasis patients after treatment with ivermectin.

The role of macrophages in the killing and elimination of microfilariae (mf) was studied immunohistologically in 14 lymph nodes from 10 patients with generalized onchocerciasis 20-68 h after treatment with a single oral dose of 150 microg/kg ivermectin. Mf with signs of damage at light microscopical level were surrounded by a cellular infiltrate comprising macrophages, eosinophils and neutrophils, whereas light microscopically intact mf mostly showed no cellular reaction. Resident mature macrophages expressing the CD 68 epitope usually neither migrated nor attached to damaged mf, especially on the first and second day after ivermectin treatment. However, many young invading macrophages labelled for the L1 protein (antibodies 27 E 10, MAC 387, S 36.48 and 8.5C2) were found within the cellular infiltrate around damaged mf and in adherence to the mf in all lymph nodes after ivermectin treatment. Free L1 protein was observed on the cuticle of the mf. The attacking macrophages contained increased amounts of the enzymes lysozyme, alpha-1-antichymotrypsin and alpha-1-antitrypsin compared to resident macrophages. Free enzymes were found on the cuticle of the mf and around them, indicating a role of these enzymes in the inflammatory reaction to the parasites. The attacking macrophages were strongly labelled for human HLA-DR and they showed further an increased expression of the complement receptors CR1 (CD 35) for C3b and CR3 (CD 11b) for C3 bi in comparison to resident macrophages and thus were considered as activated macrophages. Rarely fragments of mf were seen within multinuclear macrophages. We conclude that young activated macrophages play a major role in the elimination of mf transported to the regional lymph nodes after ivermectin treatment. The immunohistological findings are in accordance with the assumption that these activated macrophages together with granulocytes contribute to the killing of the damaged mf. They also help to limit the damage of the host tissue by release of alpha-1-antichymotrypsin and alpha-1-antitrypsin.

Differential Effects on HIV-1 Gene Regulation by EBV in T Lymphocytic and Promonocytic Cells Transduced to Express Recombinant Human CR2 (CD21)

A panel of human hematopoietic cell lines was genetically engineered to express recombinant complement receptor 2 (CR2 or CD21), which is also the Epstein–Barr virus (EBV) receptor. The panel was composed of SupT1, J1.1, and U1.HIV cells. The latter is a promonocytic cell line, whereas the other two are T lymphocytic cell lines. J1.1 and U1.HIV cells are latently infected by human immunodeficiency virus type 1 (HIV-1). These three cell lines were transduced with a murine leukemia virus (MLV)-based retroviral vector system. CR2 was efficiently and consistently expressed on the cell membranes, conferring enhanced susceptibility to EBV infection. The efficient expression of recombinant CR2 in cell lines of hematopoietic origin allowed for study of the interaction between EBV infection and HIV-1 gene regulation in suitable cell-culture models. The effects of EBV and HIV-1 coinfection results were cell-type dependent. In the two T lymphocytic cell lines, HIV-1 expression was rapidly and persistently down-regulated by EBV. Conversely, in the promonocytic cell line U1.HIV-CR2, HIV-1 expression was transiently enhanced by EBV. The EBV and HIV-1 coinfection result in U1.HIV-CR2 cells is potentially important, as the activation of HIV-1 gene expression in monocyte-like cells may play a crucial role in the mechanism of CD4+T cell depletion by apoptosis. Therefore, the U1.HIV-CR2 cell line may represent a useful cell-culture system to study the synergism between EBV and HIV-1 in inducing apoptosis in primary CD4+T cells.

Expression of complement receptors and regulatory proteins on alveolar CD4+ lymphocytes from human immunodeficiency virus-1 infected individuals.

Several lines of evidence suggest a dysregulation of the complement system in human immunodeficiency virus-1 (HIV-1) infected patients. The aim of this study was to elucidate whether CD4+ alveolar lymphocytes from HIV-1 infected patients show a loss of complement regulatory proteins that would render these cells susceptible to antibody-dependent complement-mediated cytotoxicity. We investigated the expression of complement regulatory (CD46, CD55, CD59) and complement receptor (CR1, CR2, CR3, CR4) proteins on alveolar cells by flow cytometry. Cells were obtained by bronchoalveolar lavage from 17 HIV-1 infected and 12 HIV-1 negative individuals. Expression of adhesion molecules (leucocyte functional associated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1)) and CD30 were evaluated in patient subgroups. In addition, interleukin (IL)-1beta, tumour necrosis factor alpha (TNF-alpha), IL-4 and interferon gamma (IFN-gamma) concentrations were measured in supernatants of alveolar cells. We found a significantly reduced expression of CD46 and CD59 on CD4+ alveolar lymphocytes from HIV-1 infected individuals, whereas the expression of CR3, CR4, ICAM-1 and CD30 was increased. IL-1beta and TNF-alpha concentration in supernatants of alveolar cells was augmented in HIV-1 infected patients, but did not correlate with the expression of surface molecules. IFN-gamma concentration was also increased and showed an inverse relationship to the surface expression of CD30 on CD4+. Our data suggest that in human immunodeficiency virus-1 infection an increased level of activation is associated with a diminished expression of complement regulatory proteins on CD4+ alveolar lymphocytes. This phenomenon might contribute to the depletion of CD4+ lymphocytes and the local immunodeficiency in the pulmonary compartment.

Mouse complement receptors type 1 (CR1;CD35) and type 2 (CR2;CD21): expression on normal B cell subpopulations and decreased levels during the development of autoimmunity in MRL/lpr mice.

Human complement receptors type 1 (hCR1;CD35) and type 2 (hCR2;CD21) are expressed on B lymphocytes at specific stages during differentiation and activation. These receptors play critical roles in the immune response to T-dependent Ags in addition to germinal center formation. Expression of both hCR2 and hCR1 is decreased on B lymphocytes of patients with systemic lupus erythematosus (SLE). We have studied the expression of mouse CR2 and CR1 on normal populations of mouse B lymphocytes in BALB/c mice. Our results demonstrate that expression of these receptors in the normal state closely parallels that of hCR2. During bone marrow development, expression is first detected on low B220/high IgM cells, demonstrating that complement receptors appear after central tolerance mechanisms are completed. In the splenic microenvironment the highest levels of receptor expression are found on marginal zone B lymphocytes. Mouse CR2 and CR1 are also found on peritoneal B1a and B1b cells in addition to IgA+ Peyer's patch B cells. Activation of splenic B cells under Th2 conditions results in a marked decrease in receptor expression. To determine whether the patterns of receptor expression also parallel those found in human disease, we studied the MRL lpr/lpr (MRL/lpr) model of SLE. Interestingly, we found an early decrease in complement receptor expression that is progressive and first detectable before major clinical manifestations of nephritis. We hypothesize that the early decrease in complement receptor expression such as that demonstrated by MRL/lpr mice plays an important role in the pathogenesis of murine and perhaps human SLE.

Outside-in signaling by lipopolysaccharide through a tailless integrin.

Ligand binding to integrins activates intracellular signaling pathways that coordinate and regulate a variety of cellular responses. There is evidence to suggest that the cytoplasmic tails play a key role in several of these signaling events. We sought to determine whether the beta2 integrin complement receptor type 3 (CR3; CD11b/CD18), a receptor for LPS, could initiate an intracellular signal in the absence of its cytoplasmic domains. Expression of full length CR3 in a Chinese hamster ovary-K1 fibroblast line enabled serum-independent translocation of nuclear factor-kappaB in response to binding LPS. Unexpectedly, a cell line expressing a mutated form of CR3 deficient in the cytoplasmic domains was also competent for transmitting a signal in response to LPS. In contrast, phagocytosis of whole Gram-negative bacteria and iC3b-coated erythrocytes took place only with a full length receptor. Thus, while full length CR3 is necessary for productive phagocytic signals, LPS activation does not require the cytoplasmic domains. CR3 may function to activate cells by presenting LPS to a downstream signal transducer.

Stimulation of Intracellular Ca2+ Levels in Human Neutrophils by Soluble Immune Complexes

Soluble immune complexes bind to unprimed neutrophils and generate intracellular Ca2+ transients but fail to activate the NADPH oxidase. Following priming of the neutrophils with either tumor necrosis factor alpha or granulocyte-macrophage colony-stimulating factor, stimulation of the cells with the soluble immune complexes leads to an enhanced Ca2+ signal and significant secretion of reactive oxidants. The enhanced Ca2+ signal observed in primed neutrophils results from the influx of Ca2+ from the external environment and is partly sensitive to tyrosine kinase inhibitors. This is in contrast to the Ca2+ signal observed in unprimed neutrophils, which arises from the mobilization of intracellular stores. When the surface expression of FcgammaRIIIb on primed neutrophils was decreased either through incubation with Pronase or phosphoinositide-specific phospholipase C, the extra enhanced Ca2+ mobilization seen in primed cells was significantly lowered, while the initial rise in intracellular Ca2+ was unaffected. Depletion of FcgammaRIIIb had no significant effect on the Ca2+ transients in unprimed neutrophils. Cross-linking FcgammaRII, but not FcgammaRIIIb, induced increases in intracellular Ca2+ in unprimed neutrophils, while cross-linking either of these receptors increased Ca2+ levels in primed neutrophils. The FcgammaRII-dependent intracellular Ca2+ rise in primed cells was unaffected by incubation in Ca2+-free medium, whereas the FcgammaRIIIb-dependent transient was significantly decreased when Ca2+ influx was prevented in Ca2+-free medium supplemented with EGTA. Cross-linking either FcgammaRII or FcgammaRIIIb in primed or unprimed cells failed to stimulate substantial levels of inositol 1,4,5-trisphosphate production. These results indicate that following stimulation of primed neutrophils with soluble immune complexes the enhanced Ca2+ mobilization observed is the result of a functional activation of the glycosylphosphatidylinositol-linked FcgammaRIIIb.

Altered expression of IgG and complement receptors indicates a significant role of phagocytes in atopic dermatitis

Background: Strict dietary precautions against allergic sensitization may benefit a group of predisposed children. Objective: To develop new strategies for identifying these children, a better understanding of the processes that initiate sensitization and regulate and perpetuate the inflammatory response is needed. Methods: We measured the expression of the receptors for the constant (Fc) region of IgG (FcγRI, FcγRII, and FcγRIII) and that for the complement fragments C3b and C3bi (CR1 and CR3) in neutrophils and monocytes from 39 children with atopic dermatitis, 17 disease control patients with acute infections, and 17 healthy control subjects. The capacity of phagocytes to produce reactive oxygen species was also determined. To find the best way of discriminating the patients with atopic dermatitis from control subjects, a stepwise logistic binary regression model was made. Results: The stepwise logistic regression analysis was based on differences in individual receptor expression between the study groups. Because acute infections strongly affected receptor expression in both neutrophils and monocytes, to avoid diagnostic bias, children with acute infections were excluded from the analysis. The combination of the receptors CR1 in neutrophils and FcγRI and FcγRII in monocytes was the best indicator of atopic dermatitis. A significant correlation between the expression of CR1 in neutrophils and in monocytes, as well as reactive oxygen species production of phagocytes, and the severity of the eczema was detected. Conclusions: These results suggest that a distinct receptor profile of phagocytic cells can be characterized in patients with atopic dermatitis, providing a new direction to the search for early identification of children predisposed to allergic sensitization. (J Allergy Clin Immunol 1997;99:707-13.)

Normal Expression and Function of CR3 (CD11b/CD18) on Neonatal Neutrophils(PMN) and Monocytes. • 1295

It is well known that neonates are more susceptible to bacterial infections, probably secondary to immaturity of the immune system. Previous studies have shown that neonatal PMN express less membrane and cytoplasmic CR3(iC3b-receptor) than adult PMN, and it has been suggested that this may render neonatal PMN deficient in their ability to kill iC3b-opsonized microorganisms. The current investigation examined PMN and monocytes from cord blood versus adult blood for CR3 expression and function. CR3 was measured with unseparated leukocytes using 3-color flow cytometry in which monocytes and PMN were defined as CD14+CD15- and CD14-CD15+, respectively. Although it was confirmed that neonatal PMN bore less CR3 than did adult PMN, neonatal PMN were more heterogenous, with some PMN expressing low amounts of CR3 and other PMN expressing higher adult levels of CR3. As the result of higher total PMN counts in cord blood versus adult blood (6.7 vs. 3.7 × 103/cu.mm, n=11), there were actually similar numbers of PMN in cord blood and adult blood expressing high amounts of CR3 per cell (4.7 vs. 3.7 × 103). A similar finding was made in the comparison of cord blood and adult blood monocytes for levels of CR3 expression (4.5 vs. 1.1 × 103 total monocytes; 1.87 vs. 1.0 × 103 monocytes expressing high CR3). Another flow cytometry assay was used to assess the ability of PMN and monocytes to generate a CR3-dependent respiratory burst in response to particulate β-glucan as a function of the level of CR3 per cell. The magnitude of such CR3-specific bursts was found to correlate directly with levels of CR3 expression with both PMN and monocytes. With cells expressing comparable amounts of CR3, there was no difference between neonatal and adult PMN or monocytes in this CR3 function. In conclusion, these data suggest that there is no numerical or functional deficiency of CR3 on neonatal PMN or monocytes that explains the higher susceptibility of newborns to bacterial infections. Supported by NIH grant AI-27771

Interleukin-10 down-regulates oxidative metabolism and antibody-dependent cellular cytotoxicity of human neutrophils.

The authors investigated the ability of interleukin-10 (IL-10) to modulate some constitutive or interferon-gamma (IFN-gamma)-enhanced activities of human neutrophils. An 18h culture of neutrophils with IL-10 dose-dependently down-regulated their capacity to produce O(2)- and lucigenin-amplified chemiluminescence in response to n-formyl-methionyl-leucylphenyl-alanine (FMLP). Furthermore, treatment of neutrophils with IL-10 decreased in a dose-dependent fashion, their capacity to lyse antibody-coated sheep erythrocytes. Membrane expression of Fc gamma RI, Fc gamma RII, Fc gamma RIII, CR1, CR3 and Fc gamma R- and CR-mediated phagocytosis were not modified by the cytokine. Culture of neutrophils with IFN-gamma (100 U/ml) did not modify their Fc gamma R- and CR-mediated phagocytosis, but significantly up-regulated Fc gamma RI and CR3 membrane expression as well as their oxidative metabolism and antibody-dependent cellular cytotoxicity (ADCC). When IL-10 and IFN-gamma were added simultaneously to neutrophil culture, IL-10 dose-dependently reduced IFN-gamma-induced increase of CR3 expression, O(2)- production (in response to both FMLP and phorbol 12-myristate 13-acetate, or PMA) and ADCC, but did not change Fc gamma RI expression on phagocytes. These results demonstrate that IL-10 is a significant neutrophil deactivator and provide new information on the role of IL-10 in the regulation of neutrophil-mediated inflammatory processes.

Characterization of complement receptor type i and type 3 mrna expression on polymorphonuclear leucocytes (neutrophils) in gingival crevicular fluid from periodontitis-affected patients

A previous study has demonstrated that complement receptors on the surface of polymorphonuclear leucocytes (neutrophils) in gingival crevicular fluid significantly increased compared with those in autologous peripheral blood obtained from periodontitis-affected subjects. The present study attempted to determine the mRNA levels of complement receptor types 1 and 3 (CR1, CR3) on neutrophils in gingival crevicular fluid using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. Gingival crevicular fluid samples were obtained from 11 adult periodontitis patients by gingival crevicular washing, and venipunctured peripheral blood was used as a control. RT-PCR analysis was performed using the primer sets for CR1, CR3 and β-actin. Digoxigenin-labelled RNA probes were synthesized from RT-PCR products for in situ hybridization. Both CR1 and CR3 mRNA levels relative to β-actin were significantly lower in crevicular fluid neutrophils than in peripheral blood neutrophils (crevicular fluid-CR1: 32.75±22.93%, peripheral blood-CR1: 65.30±43.25%, p < 0.005; crevicular fluid-CR3: 9.09 ± 5.34%, peripheral blood-CR3: 30.14 ± 18.80%, p < 0.005). In in situ hybridization, a greater majority of neutrophils showed positive CR1 and CR3 mRNA expression, while only a few neutrophils showed positive signals in gingival crevicular fluid. Data in the present study suggest that increased expression of complement receptors on the neutrophil cell surface appears to be unrelated to de novo synthesis.

Attenuation of Changes in Leukocyte Surface Markers and Complement Activation With Heparin-Coated Cardiopulmonary Bypass

Background. The inflammatory response induced by cardiopulmonary bypass can result in severe organ dysfunction in some patients. This postperfusion response is caused mainly by contact between blood and the foreign surface of the cardiopulmonary bypass equipment and includes adhesion of leukocytes to vascular endothelium, which precedes a series of events that mediate inflammatory damage to tissues. Methods. Low-risk patients accepted for coronary artery bypass grafting were randomized to operation with the cardiopulmonary bypass surface either completely heparin coated (Duraflo II) or uncoated. There were 12 patients in each group. Blood plasma sampled during cardiopulmonary bypass was analyzed for complement activation (C3bc and terminal SC5b-9 complement complex) and neutrophil activation (lactoferrin and myeloperoxidase). In addition, neutrophils, monocytes, and platelets were counted, and the expression of surface markers on the neutrophils and monocytes (complement receptor [CR] 1, CR3, CR4, and L-selectin) and on the platelets (P-selectin and CD41) was quantified with flow cytometry. Results. Clinical and surgical results were similar in both groups. In the group with the heparin-coated surface, the formation of the terminal SC5b-9 complement complex was significantly reduced, and the counts of circulating leukocytes and platelets were significantly less reduced initially but were higher at the end of cardiopulmonary bypass compared with baseline. Also, the expression of CR1, CR3, and CR4 was significantly less upregulated and the L-selectin, significantly less downregulated on monocytes and neutrophils. Conclusions. We conclude that heparin coating reduces complement activation and attenuates the leukocyte integrin and selectin response that occurs when uncoated circuits are used. (Ann Thorac Surg 1997;63:105–11)

A role for CR2 in FDC-B cell interactions.

If animals lack C3 or if C3 is destroyed by cobra venom factor, antibody responses are dramatically depressed1–5. Treatment of animals with mAb against CR2 or CD19, which are part of the CD19, CR2 TAPA-1 complex on B cells, also result in dramatically depressed antibody responses6–8. Furthermore, if animals are treated with a soluble construct of CR2, which will bind C3 fragments, the ability to mount a humoral response is markedly suppressed’. In addition, a recent study of CR2 knockout mice revealed that B cell expression of CR2 is required for immune responses to T-dependent antigens9. Furthermore, complement markedly lowers the threshold at which B cells respond to antigen and this effect may be attributable to the co-ligation of CR2 to BCR via the association with C3b-associated antigen10,11. It is also known that cross-linking of CR2 on the B cell by multiple C3b fragments on a carrier renders B cells more easily stimulated by mitogens including anti-μ12,13. These results suggest that CR2 is associated with an important signaling mechanism which is involved when B cells proliferate and differentiate into antibody forming cells (AFC).