Immune complex binding to erythrocyte-CR1 (CD 35), CR1 expression and levels of erythrocyte-fixed C3 fragments in SLE outpatients

Erythrocytes (E) from a cross-sectional group of 22 outpatients with systemic lupus erythematosus (SLE) and/or mixed connective tissue disease (MCTD), the majority without active disease (n = 14), were analyzed for CR1 antigen expression and capacity to bind complement opsonized, radiolabelled immune complexes (IC). Furthermore, E-bound C3 fragments and the plasma C3d concentration were determined. E-bound C3b/iC3b fragments were not elevated in patients with SLE, whereas E from 11 out of 22 SLE patients had increased C3d levels which correlated with the plasma C3d concentration (Rs 0.73, p less than 0.001). E-fixed C3d fragments did not affect the binding of Mab or preopsonized IC to E-CR1 and were not correlated with disease activity or medical treatment. Antigen expression of E-CR1 measured by ELISA or agglutination showed positive correlation with the IC binding capacity of E-CR1 (Rs 0.92 and 0.72 respectively, p less than 001). The IC binding capacity of E-CR1 from SLE patients was significantly reduced (p less than 0.005), whereas the antigen expression of CR1 (ELISA) on E from the patients did not differ from that of E from healthy donors (p greater than 0.1). E-CR1 antigen was measured by Mab reacting with an epitope outside the IC-binding site of E-CR1. E-CR1 antigen expression or IC binding showed no correlation either with disease activity or prednisolone treatment. However, 4 og 5 patients with MCTD and 4 of 5 patients receiving Imurel were found to have low E-CR1 expression and capacity to bind IC. Thus, measurement of antigenic E-CR1 in a cross-sectional group of SLE outpatients by use of Mab reacting with an epitope outside the ligand-binding region of CR1 did not reveal a significantly reduced CR1 expression. However, an assay for CR1-mediated IC binding showed a clearly reduced E-CR1 function.     

Nonopsonic binding of Mycobacterium tuberculosis to human complement receptor type 3 expressed in Chinese hamster ovary cells.

Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis is likely important in the establishment of a primary infection in the lung. M. tuberculosis binds to a variety of phagocyte receptors, of which the mannose receptor and complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a beta2 integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tuberculosis H37Rv to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesized complement, which are inherent in studies with mononuclear phagocytes. The surface expression of CD11b and CD18 was assessed by immunofluorescence, immunobead binding, flow cytometry, and immunoprecipitation with anti-CD11b and anti-CD18 monoclonal antibodies (MAbs). The functional activity of the surface-expressed CD11b/CD18 (CR3) heterodimer was confirmed by rosetting with C3bi-coated microspheres. We found that M. tuberculosis bound four- to fivefold more avidly to CR3-expressing CHO cells than to wild-type cells and, importantly, that this binding was at similar levels in the presence of fresh or heat-inactivated human or bovine serum or no serum. In contrast, Mycobacterium smegmatis bound poorly to CR3-expressing CHO cells in the absence of serum, but after opsonization in serum, binding was comparable to that of M. tuberculosis. The binding of M. tuberculosis to the transfected CHO cells was CR3 specific, as it was inhibited by anti-CR3 MAbs, particularly the anti-CD11b MAbs LM2/1 (I domain epitope) and OKM1 (C-terminal epitope), neither of which inhibit C3bi binding. MAb 2LPM19c, which recognizes the C3bi-binding site on CD11b, had little or no effect on M. tuberculosis binding. The converse was found for the binding of opsonized M. smegmatis, which was strongly inhibited by 2LPM19c but unaffected by LM2/1 or OKM1. CR3-specific binding was also evidenced by the failure of M. tuberculosis to bind to CHO cells transfected with an irrelevant surface protein (angiotensin-converting enzyme) in the presence or absence of serum. We conclude that the binding of M. tuberculosis H37Rv to CR3 expressed in CHO cells is predominantly nonopsonic and that the organism likely expresses a ligand that binds directly to CR3.     

Inhibition by monoclonal anticomplement receptor type 1 on interactions between senescent human red blood cells and monocytic-macrophagic cells

The effect of different murine monoclonal antibodies (Mab) specific for the glycoprotein complement receptor type 1 (CR1), type 2 (CR2), and type 3 (CR3) on the adhesion to and on the phagocytosis of human senescent red blood cells (S-RBC) by monocytes or by monocyte-derived macrophages (M phi) was investigated. Murine Mab anti-CR3 (anti-Leu 15 and OKM1) were found to inhibit, in the same order of magnitude, on one hand, the Fc receptors (FcR)-dependent rosetting and phagocytosis, and, on the other hand, the S-RBC rosetting and phagocytosis by adherent monocytes. Thus, the specific involvement of the CR3 epitopes recognized by Mab anti-Leu 15 or by OKM1 in the interactions between S-RBC and monocyte/macrophage could not be demonstrated. Murine Mab anti-CR1 was found to be a significant inhibitor of binding to and of phagocytosis of S-RBC (but not of young [Y] RBC) by monocytes or M phi, whereas Mab OKM5 carrying the same isotype as Mab anti-CR1, but a different specificity, was devoid of any significant inhibitory effect. Furthermore, Y-RBC or S-RBC opsonized with Mab anti-CR1 did not form FcR-dependent rosettes and were not internalized by monocytes; in addition, preincubation of phagocytes with Mab anti-CR1 did not inhibit FcR-dependent rosetting and phagocytosis. These results suggest that the effect of anti-CR1 is mediated through a specific binding to CR1 and not through an FcR blockade. As the role of specifically bound IgG on phagocytosis of human S-RBC by macrophages has previously been demonstrated by several authors, the present study suggests that monocyte-macrophage complement receptor type 1 may act in synergy with Fc receptors in the recognition of S-RBC by macrophages. It is shown in addition that the tripeptide Arg-Gly-Asp, identical to the region of iC3b recognized by CR3 and by several adhesion-promoting receptors that are structurally similar to CR3, such as fibronectin or vitronectin, is a significant inhibitor of the binding to and the phagocytosis of S-RBC by monocytic-macrophagic cells.     

Treatment with anti-CR3 antibodies ED7 and ED8 suppresses experimental allergic encephalomyelitis in Lewis rats

Experimental allergic encephalomyelitis (E AE) is an inflammatory disease of the central nervous system (CNS). Among the leukocytes which infiltrate the CNS during EAE, numerous macrophages are present. These macrophages are thought to play a crucial role in the generation of tissue damage and attendant neurological deficits. The mechanism by which the macrophages migrate across the blood-brain barrier is not yet clear. Membrane proteins involved in macrophage adherence to the endothelium include the CD11b/CD18 integrin, also known as the type 3 complement receptor (CR3). In this study we show that two monoclonal antibodies (mAb) ED7 and ED8 are directed against rat CR3. In addition, these mAb reduce recruitment of myelomonocytic cells towards thioglycollate induced peritonitis by 15-33%. This indicates that both ED7 and ED8 interfere with an epitope on CR3, which is involved in recruitment of phagocytes towards inflammatory lesions. Intravenous injection of ED7 and ED8 suppressed clinical signs of EAE. MRC OX-42, which also recognizes CR3, did not reduce thioglycollate-induced phagocyte recruitment into the peritoneum, and had no effect on EAE. These findings suggest that CR3 plays a role in the recruitment of macrophages towards the inflamed CNS of EAE animals, and confirm the role of macrophages in the generation of clinical signs of EAE. Involvement of CR3 in other phagocyte immune functions during EAE is discussed.     

Recognition of binding sites on Candida albicans by monoclonal antibodies to human leukocyte antigens.

Candida albicans exhibits examples of human molecular mimicry, including receptors resembling human steroid receptors and human complement receptor (CR)-like receptors that bind the complement fragments C3d and iC3b. To further characterize the epitopes of the Candida human CR-like molecules, a panel of monoclonal antibodies (MAbs) against epitopes within the human CR3 was used. MAbs Mo1, M1/70HL, and 7C3 bound to the germ tube, as assessed by immunofluorescence. MAbs Leu15, 60.1, and 95G8 did not bind. Miscellaneous MAbs against other antigens on human leukocytes (B2, 3D9, and OKT4) did not bind. However, MY9, which recognizes a monocyte antigen, bound extensively to the germ tube. The binding of certain anti-CR3 MAbs suggested limited identity between the Candida CR3-like receptor and the human CR3. The binding of MY9 identified an antigen recognized by a MAb to a human cell antigen not previously known to exist on C. albicans.     

Systemic suppression of human peripheral blood phagocytic leukocytes after whole-body UVB irradiation.

We examined systemic effects of whole-body UVB irradiation on human peripheral blood phagocytes. We found that 24 h after a single erythemal dose of UVB radiation two phagocyte functions, adhesion and phagocytosis, were reduced by 50%. This functional suppression was accompanied by a significant decrease in the expression of complement receptors (CR1 and CR3) and IgG Fc receptors (FcRII and FcRIII). The greatest reduction (47%) was observed in CR3, which is important for both adhesion and phagocytosis. A kinetic analysis showed that both CR1 and CR3 levels started to decrease 15 min after the UVB exposure, reaching the lowest levels at 4.5- and 24-h time points, respectively. The down-modulation of CRs after whole-body UVB exposure was not due to a defective receptor synthesis or translocation from internal stores to plasma membrane because the maximal CR levels in stimulated cells were not affected by UVB. No change in the serum soluble ICAM-1 was detected after UVB, which rules out CD1 1b epitope masking by sICAM-1. UVB did not release low-receptor-density myeloid progenitor cells from storage pools into circulation. Interleukin 10, a mediator of UVB-induced immunosuppression, was unable to modulate CR expression in vitro. When seven suberythemal whole-body UVB exposures were given repeatedly within 2 weeks, a significant decrease in CR expression was seen, which was greatest after three irradiations. Our data suggest that an exposure to UVB has systemic effects in humans which, possibly due to the down-modulation of preexisting cell-surface receptors, suppress some important functions of circulating phagocytic cells.     

Human liver Kupffer cells express CR1, CR3, and CR4 complement receptor antigens. An immunohistochemical study

The expression of complement receptor antigens by human Kupffer cells (KC) was investigated by immunohistochemical techniques in seven normal human liver biopsies. Polyclonal and monoclonal antibodies were revealed by double labeling of cells using indirect immunofluorescence and immunoenzymatic techniques or by using double immunoenzymatic techniques. In most experiments, one antigen was revealed by streptavidin-biotin-peroxidase complexes whose reaction product was examined by light microscopy and the second antigen stained using the alkaline phosphatase antialkaline phosphatase method visualized by fluorescence microscopy using fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate filters. KC were identified using monoclonal antibody EBM11 that recognizes 100% of KC in hepatic lobules and was paired with each antibody directed against complement receptors. CR1 and CR3 (alpha- and beta-chains) were found to be the predominant receptor antigens expressed by human KC. CR4 (p150,95) was expressed on all KC, but staining with anti-CR4 monoclonal antibodies was consistently weaker than that observed with anti-CR3 antibodies. No staining of KC was observed with anti-CR2 (CD 21) antibodies. Expression of CR1, CR3, and CR4 complement receptors on KC provides the cells with an optimal capacity to bind and phagocytize particles or immune complexes coated with any type of ligands for C3 receptors.     

Regulation of the adhesion versus cytotoxic functions of the Mac-1/CR3/alphaMbeta2-integrin glycoprotein

Mac-1/CR3 functions as both an adhesion molecule mediating the diapedesis of leukocytes across the endothelium and a receptor for the iC3b fragment of complement responsible for phagocytic/degranulation responses to microorganisms. Mac-1/CR3 has many functional characteristics shared with other integrins, including bidirectional signaling via conformational changes that originate in either the cytoplasmic domain or extracellular region. Another key to its functions is its ability to form membrane complexes with glycosylphosphatidylinositol (GPI)-anchored receptors such as Fc gammaRIIIB (CD16b) or uPAR (CD87), providing a transmembrane signaling mechanism for these outer membrane bound receptors that allows them to mediate cytoskeleton-dependent adhesion or phagocytosis and degranulation. Many functions appear to depend upon a membrane-proximal lectin site responsible for recognition of either microbial surface polysaccharides or GPI-linked signaling partners. Because of the importance of Mac-1/CR3 in promoting neutrophil inflammatory responses, therapeutic strategies to antagonize its functions have shown promise in treating both autoimmune diseases and ischemia/reperfusion injury. Conversely, soluble beta-glucan polysaccharides that bind to its lectin site prime the Mac-1/CR3 of circulating phagocytes and natural killer (NK) cells, permitting cytotoxic degranulation in response to iC3b-opsonized tumor cells that otherwise escape from this mechanism of cell-mediated cytotoxicity.     

The effect of antibody isotype and antigenic epitope density on the complement-fixing activity of immune complexes: a systematic study using chimaeric anti-NIP antibodies with human Fc regions.

A systematic study has been carried out to investigate the role of immunoglobulin isotype, epitope density, and antigen/antibody ratio on the capacity of immune complexes to activate the classical and alternative pathways of human complement and for the complexes subsequently to bind to erythrocyte C3b-C4b receptors (CRI). For this purpose, a series of chimaeric monoclonal anti-NIP antibodies was used, which all shared the same combining site but had different human constant domains. Antigen epitope density was varied by coupling different numbers of NIP hapten molecules to bovine serum albumin. All three parameters affect complement fixation. In general, complement activation is better in antibody excess and at equivalence than it is in antigen excess, and better at high epitope density than at low epitope density, although the effects are variable for different immunoglobulin isotypes and for the two pathways. It has been confirmed that IgG1 and IgG3 are good activators of the classical pathway and are tolerant to variations in both epitope density and antigen/antibody ratio. IgG4 and IgA do not activate the classical pathway in any circumstances. IgG2 activates the classical pathway only at high epitope density and at equivalence or antibody excess. IgM activates the classical pathway well only at the higher epitope densities and at equivalence or antibody excess but, in addition, shows an interesting and unexpected prozone phenomenon where immune complex in antibody excess inhibits complement activation by the classical pathway. The results of the alternative pathway activation are strikingly different. IgA is by far the best activator of the alternative pathway and is relatively tolerant to epitope density and to antigen/antibody ratio. IgM, IgG1 and IgG3 do not significantly activate the alternative pathway in any circumstances. IgG2 is the best IgG subclass for alternative pathway activation but requires high epitope density and equivalence or antibody excess. Binding to CR1 in general parallels the amount of complement fixed independent to the pathway by which it is fixed. However, IgG1 and IgG3 complexes in antigen excess activate complement well but bind poorly to CR1. Nascently formed complexes seem to bind complement in a way that is similar to that bound by preformed complexes, but are then less able to bind to red cell CR1. These observations help to explain the pathogenesis of complement activation in various autoimmune and immune complex diseases such as systemic lupus erythematosus, autoimmune thyroiditis and others.     

Immunohistochemical recognition of the epidermal growth factor receptor by the h-R3 antibody in the skin of experimental animals.

The h-R3 is a humanized monoclonal antibody (Mab) that binds to the external domain of the human epidermal growth factor receptor (EGF-R). It has being used for the treatment of head and neck tumors. Since it is known that different animal species have different EGF-R amino acid sequences, it raises the handicap that, a priori, the animal species relevant for the pharmacodynamic, pharmacokinetic, and toxicologic studies of this Mab are unknown. To elucidate relevant laboratory animal species, the authors investigated the immunohistochemical recognition by h-R3 Mab of EGF-R in small skin biopsy samples obtained from NMRI nu/nu, C57Bl/6, and OF-1 mice; Sprague-Dawley rats; Hartley guinea pigs; New Zealand rabbits; and Cercopithecus aethiops and Macaca fascicularis monkeys. Additionally, three human skin biopsies were obtained from trauma victims. The immunolocalization of EGF-R in different tissue sections was performed using the avidin-biotin peroxidase complex (ABC) technique. The skin sections from different tissues were tested with the biotinylated h-R3 Mab or with a biotinylated irrelevant Mab, used as negative control. The staining intensity was qualified as:-, no staining; +, weak staining; ++, moderate staining; +++, strong staining. Macaca fascicularis monkey, New Zealand rabbit, and OF-1 mouse skins had a strong staining intensity; Cercopithecus aethiops monkey and Sprague-Dawley rat skins showed a moderate to strong staining intensity; C57Bl/6 mouse skins showed a moderate staining intensity. No staining was observed in the skins from Hartley guinea pigs and MNRI nu/nu mice. The fact that h-R3 Mab recognizes other animal EGF receptors in addition to the human EGF-R makes it possible to perform relevant pharmacodynamic, pharmacokinetic, and toxicologic studies in some laboratory animals.     

Interaction between C3 and IL-2; inhibition of C3b binding to CR1 by IL-2

We previously reported that C3 has a role in the enhancement of the IL-2 dependent proliferation of helper T cells. Because the IL-2R has a structural homology with the complement proteins, such as CR1 and CR2, we studied the possible ligand crossreactions on CR1 and IL-2-receptor, and the direct interaction between C3 and IL-2. While C3 has an enhancing effect on the IL-2 dependent proliferation of HT-2, a CR1-positive mouse T-cell line, the growth of the CTLL-16 line (CR1-negative) is not affected by C3. It has been proven that neither the insolubilized C3 nor the soluble C3b-like C3 react with the IL-2 binding epitope of the IL-2 receptor. However, using human RBC we have demonstrated that the binding of aggregated C3 to CR1 is inhibited by rIL-2, in a dose-dependent manner. When RBC were incubated with rIL-2 and FITC-labelled Fab-anti-CR1 simultaneously, there was no inhibition in the fluorescence intensity. As detected by ELISA, rIL-2 was bound to the same extent by insolubilized C3, C3b, and C3c, while C3d coat had lower binding capacity. The receptor-binding epitope of IL-2 is intact in the complex of complement proteins and rIL-2, as demonstrated by the binding of DMS1, a monoclonal antibody reacting with the receptor site of IL-2. It is strongly suggested that C3b may play a role in the growth of CR1 positive T cells.     

Increased expression of C3b and C3bi receptors on human neutrophils and monocytes induced by a glycoprotein extract from Klebsiella pneumoniae (RU41740).

RU41740 is a glycoprotein extract from Klebsiella pneumoniae with immunomodulating properties under different experimental conditions. In particular the compound is able to stimulate several functions of human phagocytes in vitro and ex vivo. Using monoclonal antibodies and flow cytometry, in this work we assessed the effect of RU41740 on surface expression of receptors for C3b (CR1) and C3bi (CR3) in human phagocytic cells in vitro. The incubation of whole blood with varying RU41740 concentrations led to a dose-dependent increase in surface expression of CR1 and CR3 on both neutrophils and monocytes when compared with control samples incubated in buffer alone. The maximal drug-induced enhancement of complement receptors was: 291% +/- 13.4% for CR1 and 265% +/- 8.5% for CR3 in neutrophils; 117% +/- 4.5% for CR1 and 98% +/- 4.1% for CR3 in monocytes. These peak effects were observed using RU41740 at a final concentration of 10 micrograms/ml and were similar to those induced by optimal concentrations of the activating compound N-formyl-methionyl-leucyl-phenylalanine (10(-7)M). Polymyxin B did not modify the RU41740-induced enhancement of CR1 and CR3 expression on phagocytes, suggesting no role for endotoxin in this activity. These results define, at least in part, the mechanism of action of RU41740 on human phagocytes in vitro and could be relevant to in vivo events during RU41740 treatment.     

Cr2, a candidate gene in the murine Sle1c lupus susceptibility locus, encodes a dysfunctional protein.

The major murine systemic lupus erythematosus (SLE) susceptibility locus, Sle1, corresponds to three loci independently affecting loss of tolerance to chromatin in the NZM2410 mouse. The congenic interval corresponding to Sle1c contains Cr2, which encodes complement receptors 1 and 2 (CR1/CR2, CD35/CD21). NZM2410/NZW Cr2 exhibits a single nucleotide polymorphism that introduces a novel glycosylation site, resulting in higher molecular weight proteins. This polymorphism, located in the C3d binding domain, reduces ligand binding and receptor-mediated cell signaling. Molecular modeling based on the recently solved CR2 structure in complex with C3d reveals that this glycosylation interferes with receptor dimerization. These data demonstrate a functionally significant phenotype for the NZM2410 Cr2 allele and strongly support its role as a lupus susceptibility gene.     

Neutrophil Fcγ and complement receptors involved in binding soluble IgG immune complexes and in specific granule release induced by soluble IgG immune complexes

We examined the effect of soluble IgG immune complex (IC) characteristics on the binding of IC to human neutrophils and IC-induced specific granule release of neutrophils via Fcγ receptors (CD16 and CD32) and complement receptors (CR1 and CR3). A set of soluble IgG IC varying in size, IgG subclass, antigen epitope density and complement (C) incorporation were formed between 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) coupled to bovine serum albumin (BSA) and chimeric mouse-human anti-NIP monoclonal antibodies (mAb) of all four IgG subclasses. High and low epitope density IC of all four IgG subclasses induced specific granule release with C, but in the absence of C only IgG1 and IgG3 IC were functionally active. The Fcγ and C receptors responsible for IgG IC-induced specific granule release and IC binding were determined using mAb specific for the ligand binding sites of CD16, CD32 and CR3, and recombinant soluble CR1. Each defined IC displayed a unique pattern of receptor preference, dependent upon subclass and antigenic epitope density. IC binding and IC-induced specific granule release was not mediated by the same receptor, or combination of receptors. High and low epitope density IgG3 IC binding and induction of specific granule release was mediated predominantly via CD16. Other IC subclasses bound differently, i.e. IgG1 IC used CD16 and CR3; IgG2 and IgG4 predominantly used complement receptors; but all three induced specific granule release via CD32. In vivo these results may translate into differential activation of neutrophils by soluble IC dependent upon their characteristics, leading to subtle nuances in the etiology, pathology and control of the immune response in IC-related diseases.     

Hydrocortisone both decreases the up-regulation of complement receptors CR1 and CR3 and the ingestion process of human granulocytes

We have studied the effect of hydrocortisone on the complement receptor expression (CR1 and CR3) of human granulocytes during the up-regulation phase and the following stable period when exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the medium alone at 37 degrees C. The receptor expression was also correlated to the C3bi-mediated phagocytosis with special reference to attachment and ingestion. The results showed that after incubation with fMLP the increased expression of CR1 and CR3 is accompanied by an increased ingestion, but not attachment, of complement-opsonized yeast particles. This increase was significantly lower with regard both to attachment and ingestion, as well as receptor expression if hydrocortisone was present during the up-regulation phase. However, the addition of hydrocortisone after the up-regulation of fMLP-treated granulocytes decreased the ingestion of particles but not the CR1 and CR3 expression. The results indicate a membrane effect of hydrocortisone that affects both the receptor mobilization and ingestion mechanism of the phagocytes.     

Binding and catabolism of aggregated immunoglobulins bearing C3b or iC3b by U937 cells.

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CR1 and CR3 of human monocyte cell line U937 on the complement-mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgG to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgG to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes.     

CR1 (CD35) and CR3 (CD11b/CD18) mediate infection of human monocytes and monocytic cell lines with complement-opsonized HIV independently of CD4.

Peripheral blood and tissue mononuclear phagocytes serve as major viral reservoirs in HIV-infected individuals. We investigated the role of complement receptors CR1 (CD35) and CR3 (CD11b/CD18) in mediating productive infection with complement-opsonized HIV-1 and HIV-2 of cultured normal human peripheral blood monocytes, the promonocytic cell line THP-1, the monocytic cell line Mono Mac 6 and the glial cell line U251-MG. Cells were infected with the HTLV-IIIB strain of HIV-1 or the LAV-2 strain of HIV-2 that had been preopsonized with fresh human normal HIV seronegative serum. Productive infection was assessed by syncytia formation, the MTT cytotoxicity assay and/or release of p24 antigen in culture supernatants. Using suboptimal amounts of virus to infect the cells, we observed a higher and earlier productive infection of the cells with complement-opsonized HIV than with unopsonized virus. The enhancing effect of complement was totally suppressed by blocking CR1 or CR3 function with F(ab)'2 fragments of anti-receptor MoAbs; while blocking of the LFA-1 antigen had no effect. The infection of monocytic cells with complement-opsonized virus occurred independently of CD4 since it was not inhibited by F(ab)'2 fragments of a MoAb against the gp120 binding site of CD4 and since infection also occurred with Mono Mac 6 and U251-MG cells, which lack expression of the CD4 antigen and of CD4 mRNA. These observations suggest that complement may mediate productive infection of cells of the monocytic lineage with 'lymphocytotropic' HIV strains independently of CD4.     

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.     

Cell-associated complement regulatory proteins and their relation to disease processes]

C3-deposition is a key step for activation of the complement system, which involves C9-mediated immunocytolysis, immunoadherence, C3 receptor-mediated phagocytosis, NK potentiation, anaphylatoxin release, and amplification of C3 activation. Foreign material is eliminated even in the preimmune stage by these complement functions. Self cells, on the other hand, must circumvent the C3-attack, so that they express complement regulatory proteins, namely C3b/C4b receptor (CR1, CD35), decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46). We herein review the properties of these regulatory proteins and discuss the relationships between disease processes and the aberrance of these regulatory proteins.