Abnormal immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with AIDS.

C3b-coated immune complexes (IC) adhere to complement receptor 1 (CR1) on human E in the circulation. E from AIDS patients have an acquired low CR1 number. To study immune adherence and IC elimination in AIDS, radiolabeled hepatitis B surface Ag/antibody complexes were injected i.v. in six AIDS patients and in 14 healthy controls. The binding of IC to E was reduced in AIDS patients (mean binding 2 min after injection: 24.9 +/- 13.3%) compared with healthy individuals (63 +/- 3.7%) (p = 0.0005). The low binding correlated directly with the number of CR1/E and to the capacity of these E to bind IC in vitro. During the first 15 min disappearance of IC was faster in AIDS patients than in normal subjects and correlated with CR1 number. Thereafter, elimination was very slow in AIDS patients, which suggested that a fraction of IC might be released back into the circulation similarly to what has been observed for C3b-coated E. When the data were analyzed with a mathematical model allowing for such release to occur, five of six AIDS patients had a high release rate compared with little or no release in normal individuals (p less than 0.001). Thus, low CR1 on E is responsible for defective immune adherence, and might determine abnormal disappearance of IC from the circulation as well.     

Distribution in clusters of complement receptor type one (CR1) on human erythrocytes

The distribution of CR1 on human E was studied using label-fracture and thin section electron microscopy. CR1 was found to be organized in clusters on unfixed cells and on cells that had been prefixed with paraformaldehyde or glutaraldehyde before labeling. The number of clusters/E ranged from 8 to 20 as estimated from the examination of freeze-fracture replicas of labeled cells. Clusters contained an average of 30 to 75 gold particles on cells from two donors which expressed 462 and 586 CR1 Ag sites/cell, as determined by flow cytometry. In thin section electron micrographs, gold complexes were seen surrounding an electron-dense material protruding from the membrane which represents compact aggregates of CR1. The maximal distance between gold particles and the membrane was 100 nm, which corresponds to the estimated length of the major allotypic form of CR1, as calculated from the primary DNA sequence of the molecule. The distribution in clusters of CR1 on the E membrane may provide the basis for an enhanced affinity of C3b-CR1 interactions on the plasma membrane of the cells and may explain the preferential binding of C3b-bearing immune complexes to E in vivo.     

Expression of complement receptor type 1 (CR1) on erythrocytes of paracoccidiodomycosis patients

Complement receptor type 1 (CR1) is a membrane glycoprotein that acts as a receptor for the C3b, iC3b and C4b fragments of complement. In primates, one function of erythrocytes is to promote safe clearance of immunocomplexes (IC) from the circulation through CR1. Theoretically, in diseases characterized by high levels of circulating IC, an erythrocyte CR1 (CR1/E) deficiency may favor IC deposition in tissues or facilitate inappropriate activation of leukocytes in the circulation. Depression of the cell immune response occurs in paracoccidioidomycosis (PCM), especially in the more severe cases, and is frequently associated with high serum IC levels. In the present study we quantified the number of CR1/E in patients with the acute and chronic forms of PCM before and after treatment and correlated it with serum IC levels and CD4+ and CD8+ T cell concentration in the peripheral blood of these patients. Patients with PCM, particularly those with active disease and who had received treatment for shorter periods of time, had low numbers of CR1/E. In addition, an increase in serum IC concentration and a reduction in the CD4+/CD8+ T cell ratio were observed. After treatment there was a significant increase in mean CR1/E number and a reduction in serum IC levels. In patients with the chronic form of the disease the CD4+/CD8+ T cell ratio tended to increase after treatment and was associated with increased CR1/E levels. These results suggest that the reduction in CR1/E observed in patients is a phenomenon acquired with the disease and that CR1 could play a role in the pathogenesis of PCM.This revised version was published online in October 2005 with corrections to the Cover Date.     

Quantitative analyses of C3b capture and immune adherence of IgM antibody/dsDNA immune complexes

We isolated the IgM fraction from the plasma of an SLE patient with high titer anti-dsDNA antibodies and prepared soluble IgM/dsDNA immune complexes (IC) that fixed C and captured sufficient C3b to bind to human E via their C3b/C4b receptor, CR1 (immune adherence, IA). We used specific 125I-labeled mAb to IgM, C3b, and IgG to measure the stoichiometries of these C-opsonized IC. They contained 10 to 60 C3b and 10 to 30 IgM per PM2 dsDNA, had no detectable IgG, and the vast majority of the C3b was bound to the IgM, and not to the dsDNA. These stoichiometries are in contrast to those we observed for comparable E-bound IC prepared with IgG anti-dsDNA antibodies (100 to 200 C3b, and 200 to 500 IgG). Our results help explain the greater lability of the IgM IC with respect to IA as evidenced by their plasma-mediated release from human E (presumably due to factor I), and confirm previous predictions of a lower density of "packing" of IgM on dsDNA, compared to IgG. The detailed stoichiometry of C3b capture by the IgM IC (typically 1.5 to 3 C3b per IgM) suggests that individual IgM molecules with multiple C3b facilitate IC binding to clusters of CR1. Finally, comparison of the IgM/dsDNA IC with other IgM IC which have been investigated with respect to C activation, and review of the proposed mechanism by which IgM activates C, suggests that the nature of the Ag plays a fundamental role in determining whether or not an IgM IC can activate C and participate in IA.     

Evaluation of the mechanisms responsible for the reduction in erythrocyte complement receptors when immune complexes form in vivo in primates

Patients with immune complex-(IC) mediated diseases frequently have low levels of CR1 on E. The present study was undertaken to determine the role of circulating IC in causing low E-CR1 levels. E-CR1 were enumerated by measuring the binding of anti-CR1 mAb (E11) and rabbit anti-CR1 antibodies (RbaCR1) to E. In addition, the distribution of CR1 among E was assessed by flow cytometry of E stained with E11 and RbaCR1 and by evaluating the binding of E11-coated fluorescent beads (E11-beads) to E. E11-beads bind to clusters of CR1 on E. Five cynomolgus monkeys (CYN) were preimmunized to bovine gamma-globulin (BGG). E-CR1 changes in these animals were assessed: 1) acutely, during the first 60 min after an infusion of BGG and 2) chronically, during daily administration of BGG infusions over 2 wk. Acutely, there was a decrease in the number of E-CR1 as measured by E11 binding to E (E11/CR1). This decrease was not attributable to occupancy of CR1 by IC because the decrease in E11/CR1 number persisted after the IC had been cleared from E. By comparing the E11/CR1 levels in arterial blood to hepatic vein blood (n = 5), or in pulmonary artery blood (n = 1), we determined that the acute decrease in E11/CR1 number did not occur whereas E circulated through liver, spleen, or lung. The decrease in E11/CR1 number required the binding of IC to E because it did not occur after BGG was infused into nonimmunized CYN (n = 2) or into a preimmunized complement-depleted CYN. The decrease in E11/CR1 number was not due to loss of CR1 from E because E11/CR1 number recovered 24 h after infusion of BGG and in addition, enumeration of E-CR1 with RbaCR1 and E11-beads did not reflect a decrease in E-CR1 number. After several daily BGG infusions there was a persistent decrease in E-CR1 levels and that decrease appeared to be mainly the result of loss of CR1 from E because the decrease was confirmed with all methods of E-CR1 measurement and because E-CR1 levels recovered only slowly after BGG infusions were discontinued. Both in vitro and in vivo IC bound preferentially to subpopulations of E, identified by their ability to bind multiple E11-beads and by their high intensity staining with the anti-CR1 antibodies E11 and RbaCR1.(ABSTRACT TRUNCATED AT 400 WORDS)     

B cell complement receptor 2 transfer reaction

The B cell C receptor specific for C3dg (CR2) shares a number of features with the primate E C receptor (CR1). Previously, we have demonstrated, both in vitro and in animal models, that immune complexes (IC) bound to primate E CR1, either via C opsonization or by means of bispecific mAb complexes, can be transferred to acceptor macrophages in a process that also removes CR1 from the E. We have now extended this paradigm, the transfer reaction, to include B cell CR2. We used both flow cytometry and fluorescence microscopy to demonstrate that IC bound to Raji cell CR2, either via C opsonization or through the use of an anti-CR2 mAb, are transferred to acceptor THP-1 cells. This reaction, which appears to require Fc recognition of IgG bound to Raji cell CR2, also leads to transfer of CR2. Additional support for the B cell transfer reaction is provided in a prototype study in a monkey model in which IC bound to B cell CR2 are localized to the spleen. These findings may have important implications with respect to defining the role of C in IC handling during the normal immune response.     

Ultrastructural localization of herpes simplex virus RNA by in situ hybridization

We studied the subcellular localization of virally encoded RNA by pre-embedding in situ hybridization, using colloidal gold as an electron-dense marker. Fibroblasts infected with Herpes simplex virus (HSV) were fixed, permeabilized, then hybridized with a biotinylated HSV DNA probe under conditions favoring DNA-RNA hybrid formation. HSV probe was localized with 5-nm streptavidin-gold conjugates. Transmission electron microscopy revealed 5-nm gold in clusters and singlets within HSV-infected cells. Formalin-fixed cells contained a mean of 4.6 clusters per cytoplasmic profile and 13.2 clusters per nuclear profile. Combined formalin-glutaraldehyde fixation increased the mean number of clusters per cytoplasmic and nuclear profile to 7.2 (57% increase) and 17.5 (33% increase), respectively. Gold clusters were frequently located in regions adjacent to the nuclear envelope but were not bound to viral nucleocapsids or endoplasmic reticulum. Labeling was unaffected by pre-hybridization DNAse treatment of cells. RNAse eliminated 87% of cytoplasmic and 97% of nuclear clusters. These findings indicate that clustered gold particles labeled viral RNA, with probable binding of multiple DNA probe molecules and/or gold particles to RNA strands. This novel pre-embedding technique may be a useful tool for ultrastructural evaluation of virus-host cell interactions.     

A soluble recombinant multimeric anti-Rh(D) single-chain Fv/CR1 molecule restores the immune complex binding ability of CR1-deficient erythrocytes

CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the alpha-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.     

Quantitative analyses of the relationship between C3 consumption, C3b capture, and immune adherence of complement-fixing antibody/DNA immune complexes

We have studied the turnover of the third component of C (C3) and capture of the major cleavage fragment of C3 produced during C activation (C3b) that occurs when soluble antibody/DNA immune complexes (IC) active C. We used the Amersham RIA kit for the minor cleavage fragment of C3 produced during C activation (C3a), and a new assay utilizing mAb to C3b to measure the fraction of active C3 in a C source after the IC activate C. These mAb, along with a mAb to human IgG, allowed us to measure IC stoichiometries. The efficiency of C3 turnover by the IC is quite high, and under conditions of Ab excess, the maximum number of IgG bound per dsDNA corresponds to 1 IgG/20 to 30 base pairs. The maximum number of C3b found in the IC corresponds to less than 1 C3b/IgG, and the vast majority of the captured C3b is bound to the IgG, and not to the DNA. We identified several IC that consumed large amounts of C3, and captured large amounts of C3b, but did not bind to human E via C3b receptors (C receptor type 1). This finding suggests that the ability of IC to bind to human E depends upon the number and distribution of captured C3b molecules and the conformation and size of the DNA Ag, which reflects the need for multivalent binding between several properly arrayed C3b and a "cluster" of C receptor type 1 on the human E membrane. IC that activate C3 but do not bind to E would presumably "escape" the E IC clearance mechanism, but could deposit in susceptible organs and tissues and play a role in the pathogenesis of SLE because of their potential to generate the inflammatory products of C activation.     

Quantitative analyses of the binding of soluble complement-fixing antibody/dsDNA immune complexes to CR1 on human red blood cells

We have used direct binding isotherm analyses to measure the association constant (Ka) and number of binding sites for the binding of prepared complement-fixing antibody (Ab)/dsDNA immune complexes (IC) to human red blood cells (RBC). In order to generalize this study we have examined the binding reaction for a number of different anti-dsDNA Ab (from systemic lupus erythematosus plasmas), complement sources, RBC donors, and dsDNA sizes. The affinity of the IC for the RBC is quite high, and the Ka values fall within a narrow range (5 to 14 X 10(10) liter/mol). Similarly, the limiting stoichiometries for the number of IC bound per RBC were between 40 and 91. The very high affinity and limiting stoichiometries both suggest that the IC bind to the RBC via multiple contacts with clusters of complement receptor type 1 (CR1). Furthermore, we have used three specific monoclonal AB (mAb) to quantitate CR1 on human RBC in the presence and absence of bound IC. One of these Ab, mAb 1B4, is blocked from binding to the RBC if IC are previously bound, and we have used this observation to verify the multivalent nature of the interaction of complement-fixing IC with CR1 on human RBC.     

Disease-associated loss of erythrocyte complement receptors (CR1, C3b receptors) in patients with systemic lupus erythematosus and other diseases involving autoantibodies and/or complement activation

Although surface membrane density of complement receptor type one (CR1) on erythrocytes (E) is probably an inherited trait among normal individuals, recent evidence from our laboratories suggests that the reduced number of CR1 per E observed in patients with systemic lupus erythematosus (SLE) results from acquired as well as genetic factors. In the present investigation, the number of CR1 per E was quantitated with 125I-monoclonal anti-CR1 and was found to vary inversely with disease activity in patients with SLE who were followed serially for as long as 14 mo. Although evidence for E surface-bound immune complexes or fixed C3b/iC3b was not obtained, periods of disease activity and low amounts of CR1 per E correlated with the presence of 100 to 800 molecules per E of fixed C3dg fragments (less than 100 C3dg per E in normal subjects). Reduced CR1 and excess fixed C3dg on E also were observed in patients with other disorders associated with complement activation, including chronic cold agglutinin disease, autoimmune hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH), Sj?gren's syndrome, and mycoplasma pneumonia. A significant negative correlation (r = -0.498) between CR1/E and fixed C3dg/E was demonstrable in 255 individual assays evaluated by regression analysis. CR1 decreased and fixed C3dg increased during active disease; the converse was obtained during remission. In patients with active SLE, both serum complement activity and E CR1 decreased, whereas fixed C3dg fragments increased. By piecewise linear regression analysis, the appearance of 100 to 400 C3dg molecules on patients' E corresponded to a 27 to 60%, reduction in the number of CR1 per E (p less than 0.0002), confirming that fixation of C3 to E was correlated with a loss of CR1. In patients with PNH, low values for CR1 were observed on moderately complement-sensitive PNH type II E in association with increased fixed C3 fragments; however, the markedly complement-sensitive PNH type III E had essentially normal amounts of CR1 and bore little fixed C3. The addition of soluble DNA/anti-DNA immune complexes to normal blood generated levels of fixed C3dg fragments on E comparable to those observed on E from patients with SLE. Kinetic experiments indicated that C3b was fixed to E during the process of immune complex binding and release from E CR1, and that this fixed C3b was subsequently degraded rapidly to fixed iC3b and more slowly to fixed C3dg without the loss of CR1 that occurs in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding of model immune complexes to erythrocyte CR1 facilitates immune complex uptake by U937 cells

The E C3b/C4b receptor (CR1) has been shown to rapidly bind large complement-fixing immune complexes (IC) both in vivo and in vitro. It has been proposed that E (RBC) CR1 act as a shuttle mechanism, binding circulating IC and transporting them to tissue macrophages, thereby preventing their deposition in target tissues. In this study we have established an in vitro model system with which to study the transfer of model IC from CR1 on the RBC surface to phagocytic cells. Aggregated IgG (AHG) was opsonized with C3b, bound to RBC CR1, and the binding of these RBC-bound IC by a human monocyte cell line (U937 cells) was examined. U937 binding of AHG from the RBC surface was complete within 2 min, whereas binding of the same AHG from solution required 30 to 60 min. Despite the difference in kinetics of binding, the total amount of IC bound by U937 cells at equilibrium was the same for RBC-bound AHG and for AHG in solution. The transfer of AHG from the RBC to the U937 cell did not require exogenous factor I and was not accompanied by binding of RBC to U937 cells or by erythrophagocytosis. Our data lend support to the hypothesis that binding of IC to RBC CR1 may facilitate the clearance of IC from the circulation by enhancing their uptake by phagocytic cells.     

Processing of C3b-opsonized immune complexes bound to non-complement receptor 1 (CR1) sites on red cells: phagocytosis, transfer, and associations with CR1

Severe anemia is a lethal complication of Plasmodium falciparum malaria, particularly in children. Recent studies in children with severe P. falciparum anemia have demonstrated elevated levels of E-bound Abs, reduced E-associated complement receptor 1 (CR1) and decay-accelerating factor (DAF), and pronounced splenic enlargement, suggesting a mechanism for E loss involving Abs, complement, and phagocytosis. Motivated by these reports, we have developed an in vitro model in which human E with Abs and complement bound to CR1, DAF, or glycophorin A are incubated with model human macrophages (the THP-1 cell line). Previous work has demonstrated that immune complex (IC) substrates bound to E CR1, either by an Ab or via C3b, are transferred to macrophages with loss of CR1. In this study, we report that IC bound to DAF or glycophorin A by an Ab linkage are also transferred to macrophages. DAF is lost from the E during the transfer of DAF-bound IC, but the transfer of CR1-bound IC does not lead to a significant loss of DAF. Using glycophorin A-bound IC, we observe competition between transfer of IC and phagocytosis of the E: a fraction (相似文献   

A kinetic study of erythrocyte-DNA/anti-DNA immune complex association and dissociation reactions in systemic lupus erythematosus

Decreased binding capacity of the erythrocyte complement receptor (RBC CR1) in systemic lupus erythematosus (SLE) may contribute to abnormal handling of circulating immune complexes in these patients. Decreased numbers of RBC CR1 have been reported in SLE, but, since binding is a function of both receptor number and receptor binding kinetics, we measured kinetic parameters for the interaction of complement (C) containing [3H]DNA:anti-DNA immune complexes (IC) with normal control (NC) and SLE RBC. Experiments were performed at five temperatures ranging from 7-37 degrees C. The parameters measured included: (1) the maximum quantity of DNA:anti-DNA:C which could bind per RBC, S; (2) the association rate constant, ka, for the binding of DNA:anti-DNA:C to RBC; (3) the dissociation rate constant, kd, for the dissociation of bound DNA:anti-DNA:C IC from RBC; (4) the steady-state constant, Kss (ka/kd); and (5) the energies of activation for association, Eaa, and dissociation, Ead. Although the relative amount of bound DNA:anti-DNA:C per RBC was significantly decreased in SLE patients compared to NC (P less than 0.001), the mean values for Kss, ka, kd, Eaa and Ead did not differ significantly between the two groups. These data suggest the following: (1) RBC CR1 binding and dissociation of DNA:anti-DNA:C are consecutive reactions resulting in steady-state concentrations of free and RBC-bound IC; (2) at steady-state times, the ratio of RBC bound to unbound DNA:anti-DNA:C are governed by kinetic factors; (3) since the binding kinetics of SLE and NC RBC are not significantly different, the decreased binding activity described by other investigators can only be due to a decreased number of CR1 per RBC; and (4) values for Eaa and Ead suggest that the rate-determining steps in IC association with and dissociation from RBC involves making and breaking of hydrogen bonds.     

Structural and functional analysis of monomorphic determinants recognized by monoclonal antibodies reacting with the HLA class I alpha 3 domain.

To identify mAb reacting with the HLA class I alpha 3 domain, 14 mAb recognizing monomorphic determinants expressed on HLA-A, B, and C Ag or restricted to HLA-B Ag were screened in indirect immunofluorescence with mouse L cells expressing HLA-B7/H-2Kb chimeric Ag. mAb CR1S63, CR10-215, CR11-115, and W6/32 were found to react with the HLA class I alpha 3 domain in addition to the alpha 2 domain. mAb Q1/28 and TP25.99 were found to react only with the HLA class I alpha 3 domain. The determinants recognized by the six mAb were mapped on the HLA class I alpha 3 domain by indirect immunofluorescence staining of L cells expressing H-2Kb Ag containing different segments of the HLA-B7 alpha 3 domain chimerized with the H-2Kb alpha 3 domain. mAb TP25.99 reacts with chimeric Ag containing the HLA-B7 184 to 199 stretch, mAb CR10-215 and CR11-115 react with chimeric Ag containing the HLA-B7 184 to 246 stretch, mAb CR1S63 and Q1/28 react with chimeric Ag containing the HLA-B7 184 to 256 stretch, and mAb W6/32 reacts with chimeric Ag containing the whole HLA-B7 alpha 3 domain. Functional analysis using human CD8 alpha-bearing mouse H-2Kb-specific T cell hybridoma cells (HTB-Leu2) showed that only mAb TP25.99 inhibited IL-2 production by HTB-Leu2 cells stimulated with L cells expressing KbKbB7 Ag. This inhibition may occur because of the spatial proximity of the determinant defined by mAb TP25.99 to the CD8 alpha binding loop and/or because of change(s) in the conformation of the CD8 alpha binding loop induced by the binding of mAb TP25.99 to the HLA class I molecule. Furthermore, mAb TP25.99 inhibited the cytotoxicity of CD8-dependent and CD8-independent CTL clones. These results indicate that mAb TP25.99 has unique specificity and functional characteristics. Therefore it represents a valuable probe to characterize the role of the HLA class I alpha 3 domain in immunologic phenomena.     

Human erythrocytes inhibit complement-mediated solubilization of immune complexes

Incubation of precipitable immune complexes (IC) with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed human E were added to human serum or guinea pig serum, binding of IC to the E occurred and IC solubilization was significantly inhibited. By contrast, SRBC did not bind IC nor inhibit IC solubilization. Because IC binding to human E is mediated by CR type 1 (CR1) we evaluated whether CR1 was responsible for the inhibition of IC solubilization. Human E were treated with trypsin or anti-CR1 mAb. Both treatments abrogated IC binding to human E but did not affect the ability of the human E to inhibit IC solubilization. Human E inhibited C activation by IC. Thus, incubation of IC in human serum caused significant activation of C3 and C5, but not C4. However, when IC were incubated in whole blood or with isolated human E and serum, C3 activation by IC was inhibited significantly. In addition, we demonstrated that the C3b generated during C activation by IC deposited on both IC and human E. Thus, human E may compete for nascent C3 generated during C activation by IC. In conclusion, human E inhibit both complement-mediated solubilization of IC and C activation by IC.     

Defective immune adherence and elimination of hepatitis B surface antigen/antibody complexes in patients with mixed essential cryoglobulinemia type II

Mixed essential cryoglobulinemia type II (monoclonal Ig/polyclonal IgG) is characterized by systemic vasculitis caused by the deposition of circulating immune reactants that include the monoclonal component. Such reactants may include immune complexes (IC) formed from exogenous Ag. IC binding to E C receptor type 1 appears to play a role in transport and buffering of such IC (immune adherence: IA). To define the mechanisms responsible for immune deposition, 7 patients with cryoglobulinemia type II (IgM kappa/polyclonal IgG) and 14 normal volunteers were injected i.v. with hepatitis B surface Ag/antibody complexes. Two minutes after injection, only 19.4% (mean) of the circulating complexes were bound to E in patients as compared with 63.1% in normal subjects. This IA correlated directly with C4 and inversely with the IgM rheumatoid factor (RF) titer. Disappearance of IC was faster in patients (mean elimination rate: 15.7%/min) than in normal subjects (9.3%). In vitro experiments demonstrated that C depletion, interference with IC opsonization by monoclonal IgM RF, and decreased binding of opsonized IC in the presence of monoclonal RF are each associated with decreased IA. These observations suggest that, in patients with cryoglobulinemia type II, monoclonal IgM RF and low C contribute to reducing IA of circulating IC that might be rapidly trapped in tissues, resulting in injury.     

Class II MHC molecules and antigen enter the same vesicles during internalization by resting B lymphocytes

Efficient presentation of Ag by a B cell to a T cell requires that Ag bind to the Ag receptor (Ig) on the B cell, after which it is internalized into an acid compartment where it is modified and returned to the cell surface in the context of class II MHC molecules. It remains uncertain whether processed Ag binds to class II which has been internalized and recycled with Ag, or to nascent class II inside the cell. To determine if cell surface class II enters the same vesicles as Ag, or is excluded during internalization of Ag which is bound to the B cell receptor, 5- and 16-nm gold particles were labeled with anti-class II and anti-Ig, respectively. Cells were incubated at 37 degrees C and internalization of these particles was observed using electron microscopy. By 10 min, 60-75% of the B cell sections contained vesicles with gold particles inside them. Between 40 and 64% of these vesicles had both 5- and 16-nm particles. Maximum internalization occurred by 30-60 min, and by 2 hr the number of small and large particles on the B cell surface became constant or increased, respectively. Both kinds of particles moved from electron-lucent to electron-dense vesicles as the incubation time increased, although a portion of the anti-class II particles remained in electron-lucent vesicles. These data clearly show that labeled, cell surface class II is not selectively excluded from Ag-containing vesicles during Ag internalization. Thus, cointernalization of Ag and class II may represent a mechanism by which processed Ag meets class II.     

Phagocytosis by mouse peritoneal macrophages plated on monoclonal antibody-coated immune complex-substrates: effects of complexes of different IgG subclasses on Fc receptor functions

Macrophages plated on immune complex-coated substrates of different mouse IgG subclasses were examined for their capacities to phagocytose sheep red blood cells (SRBC) coated with monoclonal antibodies (MAb) of various IgG subclasses. IgG2a-and IgG2b-coated substrates abrogated macrophage phagocytosis of particles coated with any of the four mouse IgG subclasses. These results were confirmed by the use of two MAb of each of the IgG2a and IgG2b subclasses, with one of the MAb specific for dinitrophenyl groups and the others for SRBC. IgG3-coated substrates reduced the macrophage uptake of IgG2a-but not IgG2b-coated particles. Rabbit IgG-coated substrates ablated the uptake of SRBC coated with all mouse IgG subclasses. Resident and thioglycollate-elicited macrophages showed similar phagocytosis reduction when plated on these immune complexes. The phagocytosis of complement-coated particles was not affected by these IgG-coated substrates. Macrophages plated on both IgG2a-and IgG2b-coated substrates showed reduced immunofluorescence staining by an anti-IgG2b Fc receptor (FcR) Ab, 2.4G2 and reduced E(IgG2a) and E(IgG2b) binding. The results show that substrates coated with various IgG subclasses can abrogate phagocytosis mediated by FcR that do not have binding specificity for the substrate-immobilized Fc ligand, and suggest that the three classes of mouse FcR co-modulate.     

Aggregation of complement receptors on human neutrophils in the absence of ligand

C3bi receptors (CR3) on human polymorphonuclear leukocytes (PMN) bind ligand-coated particles and promote their ingestion. The binding activity of CR3 is not constitutive but is transiently enabled by phorbol esters (Wright, S. D., and B. D. Meyer, 1986, J. Immunol. 136:1759-1764). Our observations indicate that the capacity of CR3 to bind ligand is tightly correlated with the degree of ligand-independent aggregation of the receptor in the plane of the membrane. Fixed PMN were labeled with anti-CR3 monoclonal antibodies and streptavidin colloidal gold before viewing in the electron microscope either en face or in thin section. On unstimulated PMN, gold particles marking CR3 were dispersed randomly. Stimulation of PMN for 25 min with phorbol myristate acetate (PMA) dramatically enhances binding of C3bi-coated particles, and the CR3 on such stimulated cells was observed in clusters containing more than six gold particles. CR3 was not aggregated over coated pits. After 50 min in PMA, the binding activity of CR3 falls, and the distribution of CR3 was again observed to be disperse. If a hydrophilic phorbol ester was washed away after a 20-min stimulation, binding activity remains elevated for at least 50 min, and CR3 remained aggregated. Thus, clustering of CR3 was temporally correlated with its ability to bind ligand and initiate phagocytosis. Unlike CR3, Fc receptors and HLA did not exhibit changes in their aggregation state in response to PMA. Treating PMN with formyl-methionyl-leucyl-phenylalanine, which enhances expression of CR3 but not its function, did not lead to aggregation of CR3. These observations suggest that a clustered configuration is a precondition necessary for binding ligand and signaling phagocytosis.