A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b

The erythrocyte type one complement receptor (E-CR1) mediates erythrocyte binding of complement-opsonized immune complexes (IC), and helps protect against random deposition of circulating IC. Two linked CR1 polymorphisms occur in binding domains, at I643T and Q981H. In Caucasians, the variant alleles (643T, 981H) are associated with low constitutive E-CR1 expression levels. This study was conducted to determine if these polymorphisms affect ligand binding, and if so, represent risk factors for the autoimmune IC disease, systemic lupus erythematosus (SLE). In an ELISA comparing relative ligand binding differences, E-CR1 from individuals homozygous for the variant residues (643TT/981HH) exhibited greater binding to C4b, but not C3b, than homozygous wild-type E-CR1. Analysis of single-binding domain CR1 constructs demonstrated that the 981H residue imparted this enhanced C4b binding. No differences were observed in the 981H allele frequency between Caucasian controls (0.170, n = 100) and SLE patients (0.130, n = 150, P = 0.133), or between African American controls (0.169, n = 71) and SLE patients (0.157, n = 67). In a subset of individuals assessed for CR1 size, excluding from this analysis those expressing at least one B allele revealed a trend for over-representation of the 981H allele in Caucasian controls (0.231 frequency, n = 26) versus SLE patients (0.139, n = 83, P = 0.089), but again no difference between African American controls (0.188, n = 24) and SLE patients (0.191, n = 34). These data suggest that the 981H residue compensates for low constitutive expression of E-CR1 in Caucasians by enhancing C4b binding. This may contribute protection against SLE.     

Co-operation between human CR1 (CD35) and CR2 (CD21) in internalization of their C3b and iC3b ligands by murine-transfected fibroblasts

CR1 and CR2 are expressed as associated proteins on the B-lymphocyte surface. To investigate their respective contributions to the internalization of C3 fragments, transfected murine fibroblasts expressing human CR1, CR2, or both CR1 and CR2 were produced. CR1- and CR1-CR2-expressing cells bound C3b and C3b-dimer whereas CR2- and CR1-CR2-expressing cells bound iC3b and C3de. In all cases, maximum binding was achieved at low ionic strength. CR1-CR2-positive cells internalized two- to threefold more C3b and 1.5-fold more iC3b than CR1- and CR2-single-positive cells, respectively. Internalization of the anti-CR1 antibody J3D3, or C3de was at the same level, in both double-transfected and single-transfected cells. Furthermore, the internalization of C3b dimer by CR1-CR2 cells was impaired in the presence of OKB7, an anti-CR2-blocking antibody, but it was not altered in CR1 cells. Taken together, these findings suggest that CR1 and CR2 collaborate to internalize C3b and iC3b proteins. We suggest that the induction of conformational changes of the ligands enhances their binding to both receptors.     

The Th1 and Th2 cytokines IFN-γ and IL-4 antagonize the inhibition of monocyte IL-1 receptor antagonist by glucocorticoids: involvement of IL-1

Monocytes express IL-1 and IL-1 receptor antagonist (IL-1Ra) in response to lipopolysaccharide (LPS). IL-1 self-induction contributes to the increase in IL-1 following LPS stimulation. LPS-stimulated IL-1 and IL-1Ra production are inhibited by glucocorticoids. In the present work we examined the regulation of IL-1Ra by Th1 cytokine IFN-γ, Th2 cytokine IL-4, glucocorticoids and IL-1 in human monocytes. We demonstrate that IL-1 contributes to LPS-induced IL-1Ra expression as shown by IL-1 blockade in LPS-stimulated monocytes using a specific anti-IL-1β antibody or recombinant IL-1Ra. Glucocorticoids inhibited IL-1β-stimulated IL-1Ra mRNA expression and protein production. Glucocorticoids inhibited both IL-1-mediated and non-mediated LPS stimulation of IL-1Ra expression. Both IFN-γ and IL-4 reversed the inhibitory effect of glucocorticoids on IL-1Ra expression and secretion. The effect of IFN-γ was blocked by pretreatment of monocytes with an anti-IL-1β blocking antibody, whereas the effect of IL-4 could not be blocked, demonstrating that IFN-γ acts through a mechanism dependent on endogenous IL-1 production, whereas IL-4 acts through an IL-1-independent one. Consistent with this finding, IFN-γ (but not IL-4) failed to reverse the inhibitory effect of glucocorticoids when stimulated by IL-1, and only IL-4 combined with IL-1 showed synergism resulting in an increase in IL-1Ra production. The differential regulation and involvement of IL-1 in the expression of IL-1Ra by IFN-γ, IL-4 and glucocorticoids sets the level of monocyte responsiveness during the Th1 or Th2 responses.     

A multidonor ELISPOT study of IL-1β, IL-2, IL-4, IL-6, IL-13, IFN-γ and TNF-α release by cryopreserved human peripheral blood mononuclear cells

Utilization of cryopreserved peripheral blood mononuclear cells (PBMCs), rather than fresh ones collected from the same donor on different dates, overcomes the variability in sensitivity of these cells to activation agents. To understand the effect of cryopreservation, frozen PBMCs from eight healthy donors were studied to release T(H)1 or T(H)2 cytokines including IL- 1 beta, IL-2, IL-4, IL-6, IL-13, TNF-alpha and IFN-gamma using ELISPOT assay. The number of spot-forming cells (SFC) was determined using three concentrations of PBMCs (5 x 10(6), 5 x 10(5) and 5 x 10(4) cells/ml). PBMCs from all eight donors were found to retain their functional capacity to release T(H)1 or T(H)2 cytokines after freezing and thawing. When PBMCs were taken in concentrations 5 x 10(6) or 5 x 10(5) cells/ml, the density of IL-1 beta-, IL-2-, IL-6- and TNF-alpha-related spots in a well for most of the donors appeared to be overly high, making SFC quantification either difficult or impossible. To the contrary, PBMCs in concentration 5 x 10(4) produced distinct and quantifiable spots. The density of spots related to IL-4 and IL-13 release appeared to be optimal for SFC quantification when PBMCs were taken in concentration 5 x 10(6) whereas in 5 x 10(5) cells/ml the spot density was very low and absent in 5 x 10(4) cells/ml concentration group. No relationship between release levels of different cytokines was found, except IFN-gamma and IL-2 cytokine indicating that cryopreserved PBMCs with a high IFN-gamma response will likely have a high IL-2 response as well. Our results indicate that a release level of one cytokine may not be reliably predicted by knowing the level of the other. This implies that it is necessary to test cryopreserved PBMCs in a broad range of concentrations to determine one, which will be optimal for producing distinct and quantifiable spots.     

Modulation of neutrophil expression of C3b receptors (CR1) by soluble monomeric human C3b

Upon incubation at 37 degrees C with purified human C3b (500 micrograms/ml), polymorphonuclear neutrophils (PMN) were found to express up to 50% more C3b receptors (CR1) than PMN incubated with buffer alone. This up-regulation of CR1, assessed by the binding of radiolabeled CR1-specific monoclonal antibody, was dependent on the dose of C3b, occurred within 10-20 min and was stable for at least 90 min. PMN incubated with C3b also demonstrated enhanced CR1-dependent binding functions, such as EC3b rosette formation and phagocytosis of EIgGC3b particles. C3b at a concentration of 500 micrograms/ml induced up to 90% increase in the attachment or the phagocytic index. However, CR1 remained unable to promote phagocytosis of EC3b intermediates. Fc receptor-mediated functions were unaffected by the treatment with C3b. The active factor was characterized as monomeric C3b and, in particular, shown to be distinct from C5a. C3b purified by anion-exchange fast protein liquid chromatography on a Mono Q column or eluted from a monoclonal anti-C3b-Sepharose retained its modulating activity, while native C3 or C3 fragments such as iC3b, C3c or C3d,g were ineffective.     

IL-1β TNF-α, and IL-2 in Peritoneal Fluid and Macrophage-Conditioned Media of Women With Endometriosis

PROBLEM : The presence of various cytokines in human peritoneal fluid has been incompletely evaluated. Changes in cytokine levels may be related to the development of endometriosis, infertility, and activation of peritoneal macrophages. This study assesses levels of IL-1β, IL-2, and TNF-α in peritoneal fluid and macrophage conditioned media of women with endometriosis. METHOD : Peritoneal fluid was collected from 51 women at the time of diagnostic or operative laparoscopy for benign gynecologic disease. Peritoneal macrophages were isolated, cultured for 24 h, and the culture media collected. IL-1β, IL-2, and TNF-α levels were determined by commercial ELISA kits. RESULTS : The mean concentration of IL-1β and TNF-α was significantly higher in macrophage conditioned media of patients with endometriosis (P < 0.02). However, there were no significant changes in peritoneal fluid cytokine levels. Peritoneal macrophage concentrations were also higher in patients with endometriosis. CONCLUSION : This study supports the concept that endometriosis is associated with activation of peritoneal macrophages, and a higher concentration of these cells. This activation is reflected by the increased levels of cytokines found in macrophage conditioned media. The absence of significant changes in peritoneal fluid cytokine levels would seem to indicate that the above derangements are not responsible for the development or progression of endometriosis.     

Polarized Secretion of IL-6 by IEC-6 Intestinal Epithelial Cells: Differential Effects of IL-1β and TNF-α

Intestinal epithelial cells (IEC) can exist as polarized cells and are capable of secreting interleukin-6 (IL-6), yet it has not been determined if this IL-6 is secreted in a polarized fashion. Using the non-transformed rat IEC-6 intestinal epithelial cell line grown on microporous membrane inserts, we have determined that these cells were capable of secreting IL-6 preferentially to the basal surface when stimulated basally with IL-1p. In contrast, stimulation of the cells with TNF-a resulted in an equal level of IL-6 secretion to the apical and basal surfaces, regardless of whether the cells were stimulated by the apical or basal route. Experiments designed to test the permeability of the IEC-6 cell layer to apically added sodium fluorescein confirmed that neither IL- 1 p nor TNF-a altered the integrity of the cell layer after three days. These results suggest that IEC may have the capacity to secrete IL-6 in different patterns depending upon the stimulation received. This would allow comunication between the IEC and lamina propria cells via basal secretion and rapid communication between IEC via apical secretion.     

Phagocytosis by receptors for C3b (CR1), iC3b (CR3), and IgG (Fc) on human peritoneal macrophages

Human peritoneal macrophages (HPM) obtained via laparoscopy were examined for the presence and functional capacity of complement and Fc receptors. Between 5 and 20 ml of peritoneal fluid containing 1-2 X 10(6) macrophages/ml was available for each study. Macrophages made up 80-95% of the cells in the fluid. Fc and C3 receptors on HPM were characterized by rosette formation with, and phagocytosis of, IgG- and C3-coated sheep erythrocytes (E). ElgG were bound by 82% and ingested by 63% of HPM, with 4-15 E ingested/HPM. The HPM formed rosettes with EC3b (56%) and EC3bi (71%) but not EC3d,g or EC3d. Antibodies to complement receptors type 1 (CR1) and type 3 (CR3) inhibited rosette formation with EC3b and EC3bi, respectively, indicating that HPM possessed separate and distinct receptors for the C3b and iC3b ligands. In 60% of the samples studied, HPM demonstrated the ability to ingest both EC3b and EC3bi, as well as ElgG. Because of the heterogeneous nature of the cells obtained in peritoneal fluid, due to their progressive change from monocytelike cells into mature macrophages, HPM were separated by 1 g velocity sedimentation into fractions of increasing maturity. They were then examined for phagocytosis via Fc and complement receptors. Fc receptor mediated phagocytosis occurred throughout the monocyte-to-macrophage maturation sequence, while the ability of HPM to ingest via CR1 and CR3 was maturation dependent, with ingestion via CR3 occurring before CR1, in a manner analogous to in vitro differentiation of monocyte-derived macrophages.     

Analysis of C3b/C4b receptor (CR1) polymorphic variants by tryptic peptide mapping

The human C3b/C4b receptor (CR1) binds the major activation and opsonic fragments of the third (C3) and fourth (C4) components of complement. CR1 is a single chain integral membrane glycoprotein widely distributed on peripheral blood cells. Four codominantly inherited allelic variants with Mrs of 160,000, 190,000, 220,000 and 250,000 have been described. To address the structural basis for this unusual polymorphism, CR1 from donors expressing three of the four allelic variants was purified from surface labeled (125I) erythrocytes by iC3-Sepharose affinity chromatography and the variants compared by tryptic peptide mapping (TPM). The TPMs of each variant contained the same major peaks and minor peak areas and were nearly identical to one another. Tryptic peptide mappings of the 190,000 Mr erythrocyte CR1, which was purified prior to iodination, were similar to those derived from surface iodinated CR1. The TPMs of erythrocyte and granulocyte CR1 from the same donor differed by a single peak of increased prominence in the granulocyte map. These results indicate a conservation in amino acid sequence for those peptides detected. In view of these data and those of other studies of the structure and genetics of CR1 and related proteins, it is suggested in this paper that the allelic variation relates to CR1, being composed of repeating amino acid sequences.     

Inefficient Binding of IgM Immune Complexes to Erythrocyte C3b–C4b Receptors (CR1) and Weak Incorporation of C3b–iC3b into the Complexes

The binding of soluble complement-reacted IgM immune complexes (IC) to erythrocyte (E) C3b–C4b receptors (CRI) and the incorporation of C3b–iC3b into solid phase IgM-IC was investigated. The optimal binding of liquid phase IgM-IC to E-CRI was obtained with IC formed at moderate antibody excess, but the binding was low (2–3%) when compared to the binding of the corresponding IgG-IC (50–60%). Solid phase IC were prepared by coming microwells with heat-aggregated bovine serum albumin (BSA) followed by incubation with rabbit IgM anti-BSA antibody. The IC were reacted with human serum at 37°C. The binding of C3b–iC3b was determined by use of biotinylated F(ab')₂ antibodies to C3b-C3c and avidin-coupled alkaline phosphatase. The incorporation of C3b–iC3b into solid-phase IgM-IC increased when increasing amounts of IgM antibody were reacted with the antigen. The binding reaction was slow, reaching a maximum after about 2 h at 37°C. The binding of C3b–iC3b to the IgM-IC was remarkably inefficient when compared to the incorporation into IgG-IC reacted with the same amounts of BSA-precipitating antibody.     

An interaction between CD16 and CR3 enhances iC3b binding to CR3 but is lost during differentiation of monocytes into dendritic cells

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC.     

Phagocytosis of Agarose Beads by Receptors for C3b (CR1) and iC3b (CR3) on Alveolar Macrophages from Patients with Sarcoidosis

Alveolar macrophages (AM) from sarcoidosis patients exhibit no detectable defect in their potential to synthesize the functional alternative and terminal pathway of complement. They also synthesize more C9 than AM from healthy controls. Various authors [4, 6] have suggested that sarcoid AM have decreased phagocytic ability. In the present work we studied whether there was any difference in C3 receptor-mediated phagocytosis of serum-treated and native agarose beads by AM recovered from patients with active sarcoidosis compared with controls, AM from seven patients with active sarcoidosis and seven healthy controls were cultured under serum-free conditions for 2, 12, 24. and 48 h. We found a significantly increased CR1 and CR3 receptor-mediated phagocytosis of native agarose heads by AM from the seven patients. CR1 and CR3 were also detected on AM directly recovered from bronchoalveolar lavage fluid using fluorescein-conjugated monoclonal anti-receptor antibodies. The percentage of AM expressing CR appeared to be increased in sarcoidosis. The reason for the enhanced phagocytosis of agarose beads by the sarcoid AM is probably the result of both increased synthesis and receptors of complement. Altered complement production and complement receptors may be important for the pathogenesis of this granulomatous disorder.     

Covalent disulfide binding of human IL-1 beta to alpha 2-macroglobulin: inhibition by D-penicillamine.

Secreted human IL-1 beta is known to have two free SH groups due to unpaired cysteines (positions 8 and 71). Alpha 2-Macroglobulin (alpha 2-M) has internal thioester bonds between cysteine and glutamate residues. Free SH groups may be generated at these alpha 2M residues through the action of proteinases, amines such as methylamine, or at a slow rate, by H2O ("aging" of alpha 2M). Thus, the possibility that IL-1 beta forms a disulfide bond with alpha 2M was investigated. 125I-labeled human rIL-1 beta (15 kDa) was incubated with fresh normal human serum or with purified alpha 2M, treated or not with methylamine. The mixtures were submitted to nondenaturing and denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography. IL-1 beta bound to commercially purified "aged" alpha 2M and to alpha 2M in methylamine-treated serum but not to native serum alpha 2M. It did not bind detectably to any other serum proteins. The addition of D-penicillamine (D-pen) during the reaction of [125I]rIL-1 beta with serum or purified alpha 2M blocked the covalent binding of rIL-1 beta to alpha 2M. [125I]rIL-1 beta was removed from alpha 2M by 2-mercaptoethanol in SDS. Thus, disulfide bonds were formed between the free SH groups on [125I]rIL-1 beta and those resulting from the cleavage of the internal thioester bonds of alpha 2M. "Cold" rIL-1 beta and a Cys71----Ser71 rIL-1 beta mutant effectively competed with [125I]rIL.1 beta for binding sites on alpha 2M. When complexes of rIL-1 beta or the mutant rIL-1 beta and alpha 2M were subjected to nonreducing SDS-PAGE and subsequent Western blot analysis, the rIL-1 beta molecules were found to be present in the alpha 2M bands in a dose-dependent manner. rIL-1 beta attached to alpha 2M in the presence or absence of D-pen showed similar biological activity in the mouse thymocyte-assay. Thus, rIL-1 beta attached noncovalently to alpha 2M is biologically active. The lack of inhibition of rIL-1 beta activity by binding to methylamine-treated alpha 2M in the absence of D-pen suggests, but does not prove, that the covalently bound rIL-1 beta is also active. We concluded that human rIL-1 beta binds to alpha 2M through the Cys at position 8 and that D-pen inhibits this binding. We speculate that this inhibitory effect may contribute to the therapeutic benefits of D-pen in patients with rheumatoid arthritis.     

Social stress enhances IL-1β and TNF-α production by Porphyromonas gingivalis lipopolysaccharide-stimulated CD11b+ cells

Psychological stress is associated with an increased expression of markers of peripheral inflammation, and there is a growing literature describing a link between periodontal pathogens and systemic inflammation. The hypothesis of the present work is that exposing mice to the social stressor, called social disruption (SDR), would enhance the inflammatory response to lipopolysaccharide (LPS) derived from the oral pathogen, Porphyromonas gingivalis. Mice were exposed to SDR for 2 h per day on 6 consecutive days. On the morning following the last cycle of SDR, mice were tested for anxiety-like behavior in the open field test and novel object test. The mice were sacrificed the following day and their spleens harvested. Spleen cells were stimulated with LPS derived from P. gingivalis in the absence or presence of increasing doses of corticosterone. Social disruption resulted in anxiety-like behavior, and the production of IL-1β and TNF-α was significantly higher in spleen cells from mice exposed to SDR in comparison to levels from non-stressed control mice. In addition, the viability of spleen cells from mice exposed to SDR was significantly greater than the viability of cells from non-stressed control mice, even in the presence of high doses of corticosterone. The use of cultures enriched for CD11b+ cells indicated that the stressor was affecting the activity of splenic myeloid cells. This study demonstrates that social stress enhances the inflammatory response to an oral pathogen and could provide a critical clue in the reported associations between stress, inflammation, and oral pathogens.     

Interaction between immune complexes and C3b receptors on erythrocytes

We studied the interaction between immune complexes (IC) and C3b receptors (CR1) on erythrocytes (E) and showed that activation of the classical complement pathway is essential for the binding of IC to CR1, that C3b inactivator (I) and beta 1H (H) are essential for the release of IC from CR1, that CR1 retain the capacity to bind IC after repeated binding and release of IC, and that on the other hand, IC lose the capacity to bind to CR1 after repeated binding and release. These results suggest a dynamic in vivo interaction between IC and CR1 on E which are supposed to transport IC to the reticuloendothelial system. CR1 on E, with a help of I and H, might make IC less harmful to the tissues in the process of releasing IC from E.