Relationship between complement activation and renal deposition of immune complexes made with IgG2a monoclonal antibodies.

Previous studies identified a single murine monoclonal IgG2a anti-dinitrophenyl antibody that, when combined with the antigen, formed immune complexes (IC) that were preferentially deposited in glomeruli. The present study examined the clearance and organ localization in Balb/c mice of expanded panels of radiolabeled IC containing murine monoclonal antibodies. The results identified a second IgG2a antibody that formed IC with a predilection for renal deposition. IC made with the two IgG2a antibodies that were preferentially deposited in the kidney were the least efficient binders of human C1q or homologous murine C3b and C4b within the IgG2a panel. These observations suggest a new model of IC-mediated renal disease initiation in which relatively weak complement activation leads to inefficient IC clearance by complement receptor-bearing circulating cells and consequent IC deposition in tissues susceptible to IC-mediated injury.

Glomerular deposition of immune complexes made with IgG2a monoclonal antibodies.

The factors that determine whether immune complexes (IC) are cleared safely from the circulation or are deposited in vulnerable tissues such as glomeruli are not well defined. To better understand how IC are handled, the present study examined the fate in vivo of three model IC preparations with different immunochemical characteristics. Radiolabeled IC were constructed with murine IgG1, IgG2a, or IgG3 anti-DNP mAbs bound to DNP-BSA, designated IgG1 IC, IgG2a IC, and IgG3 IC, respectively. The IC were infused i.v. into BALB/c mice, and clearance and tissue localization of the three IC probes were compared. The results indicate that the major portion of each IC preparation was cleared from the circulation by the liver. However, compared with the other two probes, IgG2a IC were preferentially deposited in the kidney. Histologic examination revealed the presence of IgG2a IC in glomeruli. The enhanced renal uptake of IgG2a IC could not be attributed solely to such characteristics as IC size, Ag/Ab ratio, Ab charge, or affinity. However, the preferential renal deposition of IgG2a IC was abrogated by complement depletion. Thus, enhanced renal uptake in normal mice was complement dependent. These data suggest that interactions between IC and the complement system can influence the propensity of IC to deposit in tissues susceptible to IC-mediated injury.

Ig N-glycan orientation can influence interactions with the complement system.

This study was prompted by the paradoxical observation that a pair of dinitrophenyl-specific murine monoclonal IgG2a Abs had similar monosaccharide content and yet differed in their binding to lectins. The differential lectin-binding properties were lost when the Abs were denatured, suggesting that variations in lectin binding reflected the conformational accessibility of the N-glycans rather than intrinsic differences in the lectin binding capacity of the glycans themselves. This hypothesis was supported by experiments indicating that the degree to which the N-glycans on the Abs were reactive with beta-1,4-galactosyltransferase or susceptible to peptide N-glycosidase F corresponded directly to their relative accessibility to lectins. Moreover, the relative susceptibility to these enzymes and accessibility to lectins was inversely related to the capacity of the Abs to activate the classical pathway, suggesting that the orientation of the more accessible N-glycan might inhibit C1q binding. This hypothesis was supported by evidence that enzymatic cleavage of the more accessible N-glycan resulted in enhanced Clq, C4b, and C3b deposition. Conversely, removal of the less accessible N-glycan expressed by the other Ab inhibited C1q, C4b, and C3b deposition. The respective increase or decrease in C3b deposition on the two deglycosylated Abs was magnified when complement activation was performed in factor B-depleted serum, suggesting that N-glycan conformation primarily affects the classical pathway. Collectively, these data suggest that the orientation of the N-glycan expressed on Igs can profoundly influence interaction with the complement system.

Effect of immunoglobulin variable region structure on C3b and C4b deposition.

Many of the biological activities of immunoglobulins, including interaction with the complement system, are attributed to the structure of the heavy chain constant domains. However, previous studies indicated that immune complexes formed with independently derived isotype-matched pairs of monoclonal antibodies vary with respect to their capacity to activate complement and to serve as targets for C3b and C4b deposition. The goal of the present study was to provide a structural basis for explaining how variable domains influence C3b and C4b deposition on immunoglobulins. Heavy and light chain variable domains from a pair of IgG2a antibodies previously shown to differ in terms of complement activation and C3b and C4b deposition were cloned and sequenced. The two clones utilize distinct heavy and light variable region genes and the translated amino acid sequence reveals several residues that could serve as potential targets for complement deposition which differs between the two antibodies. Molecular modeling suggests that many of the relevant differences between the two antibodies are located in solvent exposed portions of the heavy and light chain variable domains and that some of the relevant sites are located within the complementarity determining regions. Differences in antibody affinity do not provide an explanation for the previously observed role of variable domains on interactions with the complement system. These data suggest that sequence variations within solvent-exposed variable domain residues may play a key role in C3b and C4b deposition on immunoglobulins.

Expression of the CR1 receptor on human leukemia-derived CD4+ T cell lines.

The complement receptor type 1 (CR1) is a membrane glycoprotein expressed on a variety of cells including some, but not all, human T lymphocytes. The present study was designed to investigate CR1 expression on human leukemia-derived CD4+ T cell lines. The expression of CR1, as well as the complement receptor type 2 (CR2) and membrane cofactor protein (MCP), were analyzed on cells from the SUP-T1, CEM-SS, HUT-78, and MOLT-3 cell lines by flow cytometry. All four cell lines expressed CR2 and MCP, but only the SUP-T1 cell line contained CR1+ cells. Within the SUP-T1 cell line, a mean of 8.5% of the cells were CR1+. Scatchard analysis indicated that approximately 2700 CR1 molecules were expressed per CR1+ SUP-T1 cell, a value that corresponds to the quantitative expression of CR1 on normal peripheral blood T lymphocytes. Separation of CR1+ and CR1- cells within the SUP-T1 cell line by flow cytometry and subsequent reculture of the sorted cells showed that both the enriched CR1+ and the enriched CR1- cell populations returned to a mixed CR1 phenotype over time. These data suggest that SUP-T1 cells can express CR1, but only transiently. Two-color flow cytometric analysis indicated that the expression of CR1 by SUP-T1 cells did not fluctuate with the cell cycle. SUP-T1 cells were also cultured in the presence of agents known to activate T cells. Phytohemagglutinin and phorbol 12-myristate 13-acetate induced a transient increase in the percentage of SUP-T1 cells expressing CR1, compared to cells cultured in media alone. These results suggest that CR1 expression is up-regulated during T cell activation. The CR1+ SUP-T1 cell line, as well as the CR1- cell lines, may provide useful models for investigating how CR1 expression is regulated and for exploring a possible role for CR1 in the pathogenesis of AIDS.

Relationship between immune complex binding and release and the quantitative expression of the complement receptor, type 1 (CR1, CD35) on human erythrocytes.

Primate erythrocytes (E) play a central role in clearing potentially pathogenic immune complexes (IC) from the circulation. E capture circulating IC via interaction between C3b and C4b sites, generated on the IC during activation of the complement cascade, and the complement receptor, type 1 (CR1), expressed on E. IC are released from E when C3b and C4b sites on the IC are cleaved by Factor I. The goal of this study was to examine the interactions between human E and model IC in the context of quantitative variations in CR1 expression. IC were prepared by combining murine monoclonal IgG1, IgG2b, or IgG3 anti-dinitrophenyl (DNP) antibodies with DNP-bovine serum albumin. The expression of CR1 on E, obtained from eight healthy donors, was quantified by radioimmunoassay and Scatchard analysis. On the basis of quantitative CR1 expression, preparations of E obtained from different donors at various times were categorized into phenotypic groups expressing high, intermediate, or low numbers of CR1. While there was some variation in the expression of CR1 of individual donors, five of the eight donors remained within the same phenotypic group upon repeated sampling. Surprisingly, when interactions between IC and E were examined in vitro, there was no direct relationship between the number of CR1 per E and the peak magnitude of IC binding to E. When peak binding and release rates were calculated, there was a direct correlation between the number of CR1 per E and the peak binding rate of IC constructed with IgG3 antibodies (IgG3 IC). In addition, there was an inverse correlation between the number of CR1 per E and the peak release rate of IgG2b IC. There was no direct correlation between the quantitative expression of CR1 on E and the peak binding or release rates of IgG1 IC. These data indicate that the quantitative expression of CR1 can affect the interactions between IC and E, but that these interactions are also dependent upon the immunochemical properties of the IC. These findings may be relevant to the pathogenesis of diseases, including systemic lupus erythematosus and AIDS, in which E express reduced numbers of CR1.

Distribution and quantitative expression of the complement receptor type 1 (CR1) on human peripheral blood T lymphocytes.

The complement receptor, type 1 (CR1) is expressed on a variety of cell types including primate erythrocytes, phagocytic cells, and B lymphocytes. On these cells, CR1 plays a role in a diverse spectrum of biological activities including the clearance of immune complexes from the circulation, down-regulation of the complement system, recognition of complement-coated microorganisms, and cellular activation. CR1 is also expressed by some, but not all, T lymphocytes. The present study was undertaken in order to examine the distribution of CR1 on normal human T cell subsets by flow cytometry and to quantify the expression of T cell CR1 by radioimmunoassay. Data presented here indicate that, in a panel of 19 normal individuals, a mean of 9.7% of the overall peripheral blood lymphocyte population expressed CR1 and that, as assessed by two-color flow cytometry, 12.0% of CD3+, 13.0% of CD4+, and 20.0% of CD8+ cells expressed CR1. While single peaks of CR1 staining were observed within the CD3 and CD4 subsets, a biphasic pattern of staining was evident within the CD8 subset in which relatively high-intensity CR1 staining was detected within the subpopulation of "dull" CD8+ cells, whereas a lower intensity of CR1 staining was observed within the subpopulation of "bright" CD8+ cells. Duplicate analyses performed over a relatively short time frame suggested that, while the overall percentage of cells that expressed CR1 varied considerably among normal individuals, in at least some individuals the percentage of cells expressing CR1 was relatively stable, especially within the CD4 subset. In cell suspensions enriched for T lymphocytes by rosetting with sheep erythrocytes, 10.0% of the cells were CR1+ and a mean of approximately 3700 CR1 were expressed per CR1+ cell. There was no apparent correlation between the number of CR1 per T cell and the number of CR1 expressed per erythrocyte in the same blood sample. The expression of CR1 on subpopulations within the CD3+, CD4+, and CD8+ lymphocyte subsets may play a role in both normal cell function and in the pathophysiology of disease states including the acquired immune deficiency syndrome (AIDS).

Genetic resistance in experimental autoimmune encephalomyelitis. I. Analysis of the mechanism of LeR resistance using radiation chimeras.

Experimental autoimmune encephalomyelitis (EAE) is a cell-mediated autoimmune disease of the central nervous system that has been extensively studied in the rat. The Lewis rat is highly susceptible to the induction of EAE, while the Lewis resistant (LeR) rat is known to be resistant. In this paper, we demonstrate that the LeR rat, which was derived from the Lewis strain by inbreeding of fully resistant animals, is histocompatible with the Lewis strain. Radiation chimeras, a tool for distinguishing between immunologic and nonimmunologic resistance mechanisms, were utilized to analyze the cellular mechanisms involved in genetic resistance to EAE. By transplanting bone marrow cells from LeR rats into irradiated Lewis recipients, Lewis rats were rendered resistant to EAE induction. Likewise, transplanting Lewis bone marrow cells into irradiated LeR recipients rendered LeR rats susceptible. Mixed lymphoid cell chimeras using bone marrow, spleen, and thymus cells in Lewis recipient rats revealed individual lymphoid cell types and cell interactions that significantly affected the incidence and severity of EAE. Our results suggest that LeR resistance is mediated by hematopoietic/immune cells, and that cells located in the spleen appear to play a critical role in the resistance/susceptibility to EAE induction. Depletion of splenic adherent cells did not change the patterns of EAE resistance. In vivo cell mixing studies suggested the presence of a suppressor cell population in the LeR spleen preparations which exerted an inhibitory effect on Lewis autoimmune responses. Thus, the mechanism of LeR resistance appears to be different from that in other EAE-resistant animals.

Differential binding of immunoglobulin A and immunoglobulin G1 immune complexes to primate erythrocytes in vivo. Immunoglobulin A immune complexes bind less well to erythrocytes and are preferentially deposited in glomeruli.

Primate erythrocytes appear to play a role in the clearance of potentially pathogenic immune complexes (IC) from the circulation. This study was undertaken to compare the clearance from the circulation and tissue uptake of two monoclonal IC probes: one of which, IgG1-IC, was bound well by erythrocytes, the other of which, IgA-IC, was bound relatively poorly by erythrocytes. The IC probes were labeled with different iodine isotopes and infused either concomitantly or sequentially into the arterial circulation. The results indicate that, compared with IgG1-IC, IgA-IC bind less well to primate erythrocytes, are cleared from the circulation more quickly despite their smaller size, and show increased uptake in kidney and lung but decreased uptake in liver and spleen. Evidence is presented which suggests that this pattern of clearance from the circulation and systemic uptake of IgA-IC is the result of decreased binding of IgA-IC to circulating erythrocytes. These findings support the hypothesis that the primate erythrocyte-IC clearing mechanism may be critically important for the safe removal of IC from the circulation.

Complement depletion accelerates the clearance of immune complexes from the circulation of primates.

Binding of immune complexes (IC) to erythrocytes in vitro is the result of interaction between C3b sites on the IC, and complement receptors type I (CRI) expressed on primate erythrocytes. Recent evidence indicates that primate erythrocytes can also rapidly bind large, preformed IC in vivo. This study was undertaken to determine if the binding of IC to baboon erythrocytes in vivo is complement dependent and to examine the effect of complement depletion on IC clearance from the circulation. The results indicate that complement depletion in vivo reduced the binding of IC to erythrocytes. There was relatively little binding of IC to leukocytes in both the complement-depleted and complement-repleted condition. Thus, the majority of IC not bound to erythrocytes remained free in the plasma and, consequently, IC infusion during the complement-depleted state resulted in increased plasma IC concentrations. This was associated with a rapid disappearance of IC from the circulation. By contrast, in the normal or complement-repleted state, a large fraction of the IC became bound to erythrocytes during IC infusion, which resulted in lower plasma IC concentrations. Under these conditions, a more gradual rate of disappearance of IC from the circulation was observed. The relatively abrupt clearance of IC from the circulation in the complement-depleted state could not be accounted for by increased hepatic or splenic uptake. These data indicate that, in contrast to previous studies in nonprimates, complement depletion in primates results in accelerated removal of IC from the circulation. This suggests that factors such as hypocomplementemia and deficient expression of erythrocyte CRI, which are known to occur in certain IC-mediated diseases, may promote IC uptake by organs vulnerable to IC-mediated injury.

Modification of the clinical and histopathologic expression of experimental allergic encephalomyelitis by the vasoactive amine antagonist cyproheptadine.

Experimental allergic encephalomyelitis (EAE) is an autoimmune syndrome that can be induced in Lewis rats by myelin basic protein (BP) in complete Freund's adjuvant (CFA). Rats that have recovered from a primary episode of EAE display paradoxical long-term resistance to EAE reinduction by BP-CFA. Previous observations indicated, however, that clinical disease could be reinduced in convalescent rats by a concomitant secondary challenge with BP-CFA + Bordetella pertussis extract (PERT). Vascular permeability changes in the central nervous system (CNS) paralleled disease reinduction. To further probe the relationship between disease reinduction and vascular permeability, convalescent rats were treated with the vasoactive amine antagonist cyproheptadine (CYP) prior to a secondary challenge with BP-CFA + PERT. Data presented here indicate that CYP treatment results in substantial protection of convalescent rats from clinical disease reinduction by BP-CFA + PERT. CYP did not, however, prevent the development of new CNS lesions. CYP therapy also altered the clinical course of EAE induced by a primary injection of BP-CFA + PERT. In these rats, there was a delay in the onset of clinical signs as well as in the appearance of CNS lesions. Nevertheless, both CYP-treated and untreated naive rats challenged with BP-CFA + PERT eventually developed severe and usually lethal EAE. The effect of CYP on EAE induced in naive rats without including PERT in the sensitization protocol was also evaluated. In contrast to the mitigating effect of CYP on EAE induced or reinduced by BP-CFA + PERT, CYP treatment did not affect the clinical course or the development of CNS lesions in rats challenged with BP-CFA alone. Likewise, the passive transfer of EAE, mediated by mitogen-stimulated cells obtained from BP-CFA-sensitized donors, was not affected by CYP treatment. Collectively, these data indicate that CYP therapy altered the expression of EAE induced by regimens that included PERT, but did not affect EAE induced without PERT. In view of the opposing effects of PERT and CYP on vascular permeability, these data are consistent with the hypothesis that alterations in vascular permeability may play a crucial role in controlling the expression of autoimmune neurological diseases.

Factors influencing the binding of large immune complexes to the primate erythrocyte CR1 receptor.

This study was undertaken to ascertain the relationship between the size of immune complexes and their capacity to be bound by primate erythrocytes, and to examine the role of complement in the binding of very large immune complexes to erythrocytes. Preformed bovine serum albumin-rabbit anti-bovine serum albumin immune complexes were solubilized in a fivefold antigen excess, and the supernatant fluids were subjected to differential centrifugation to select immune complexes of varying size. The size profile of immune complexes was measured on isokinetic sucrose gradients. Immune complexes were incubated with erythrocytes, and unbound immune complexes were separated from erythrocyte-bound immune complexes by centrifugation on Percoll. The results indicate that large immune complexes were bound more efficiently by primate erythrocytes than were smaller immune complexes. Depletion of complement by a variety of procedures abrogated binding of immune complexes to erythrocytes. Only the erythrocytes of primates had the capacity to bind immune complexes. Baboon or rabbit sera supported immune complex binding to baboon or human erythrocytes. In contrast, human and guinea pig sera supported immune complex binding to human erythrocytes, but these sera failed to support immune complex binding to baboon erythrocytes. These data indicate that the immune complex size and the source of serum complement are important factors which influence the binding of immune complexes to primate erythrocytes.

Leukocyte subpopulations which amplify or suppress antigen-induced proliferation in Syrian hamsters.

The proliferative response of spleen cells, obtained from Syrian hamsters sensitized to hen egg albumin emulsified in complete Freund's adjuvant, is lower in magnitude than the response of draining lymph node cells. In this study, the cellular regulatory mechanisms which may lead to splenic hyporesponsiveness were examined. Although unfractionated spleen cells were not suppressive, the addition of nylon wool nonadherent normal spleen cells to sensitized draining lymph node (target) cells markedly suppressed antigen- but not mitogen-induced proliferation. Suppressor cell activity was not detected in normal lymph nodes. Suppression could be overcome by culturing splenic suppressor plus target cell mixtures in the presence of large quantities of antigen. Suppressor cell activity was radioresistant. In addition to nonadherent suppressor cells, the hamster spleen also contains an adherent cell population(s) which amplified antigen-induced proliferation. Adherent cells with amplifying activity were also present in lymph nodes. The addition of adherent cells abrogated splenic suppression of proliferation. Collectively, these data indicate that the hamster spleen contains both suppressive and amplifying leukocyte subpopulations which may be involved in the regulation of the immune response to certain antigenic stimuli.

Primate erythrocyte-immune complex-clearing mechanism.

Previous in vitro studies have shown that immune complexes (IC) that fix complement can bind to the C3b receptor on primate erythrocytes. The in vivo function of this erythrocyte receptor, however, is unknown. This study was undertaken to determine whether the binding of IC to erythrocytes in vivo might play a role in the removal of IC from the circulation. Baboons and rhesus monkeys were prepared with a catheter in the ascending aorta to infuse IC and in the abdominal aorta, renal, hepatic, and portal veins to monitor changes in binding and clearance of IC across kidney, liver, and spleen + gut, respectively. Autologous 51Cr-labeled erythrocytes were infused intravenously and allowed to equilibrate. Preformed IC (125I-labeled bovine serum albumin [BSA] rabbit anti-BSA) were then infused into the ascending aorta at a constant rate for 120 s. Blood samples were drawn at frequent intervals for 30 min from all catheters below the IC injection site. Each blood sample was then centrifuged on percoll to separate IC bound to erythrocytes from IC in plasma or bound to buffy coat cells. This resulted in an "erythrocyte fraction" beneath the percoll that contained the IC bound to erythrocytes, and a "plasma/buffy coat fraction" above the percoll that contained the IC in plasma and IC bound to buffy coat cells. Analysis of these data showed that the majority of the IC infused into the circulation rapidly became bound to erythrocytes. However, by 5 min after beginning the IC infusion, most of this IC load had been removed from the erythrocytes as they traversed the liver. In contrast, IC on erythrocytes did not deposit in kidney. The IC-bearing erythrocytes themselves were not trapped or detained by any organ. IC in the plasma/buffy coat fraction of blood were removed from the circulation but at a relatively low rate and almost entirely by the liver. These findings suggest that primate erythrocytes intercept large complement-fixing IC in the circulation causing the IC to adhere to the erythrocyte until th e IC-bearing erythrocyte traverses liver where the IC is deposited, and the erythrocyte is returned to the circulation. This primate erythrocyte-IC-clearing mechanism may be important in the protection against diseases mediated by deposition of circulating IC.

Regulation of paralytic experimental allergic encephalomyelitis in rats: susceptibility to active and passive disease reinduction.

Spleen cells obtained from Lewis inbred rats previously immunized with 50 microgram of guinea pig basic protein emulsified in complete Freund's adjuvant passively transferred paralytic experimental allergic encephalomyelitis (EAE) only following an in vitro conditioning period. The in vitro conditioning required the presence of either concanavalin A or encephalitogenic antigen. The conditioned cells caused passive paralytic EAE in recipients of 10 million viable cells. After recovery from passive paralytic EAE, animals were completely susceptible to additional attempts to induce EAE by active or passive means. Active EAE recovered rats were resistant to additional attempts to induce active EAE but were fully susceptible to passively induced EAE mediated by in vitro conditioned cells.

Genetic resistance to the induction of experimental allergic encephalomyelitis in Lewis rats. I. Genetic analysis of an apparent mutant strain with phenotypic resistance to experimental allergic encephalomyelitis.

Clinical resistance to the induction of experimental allergic encephalomyelitis was observed in a closed colony of Lewis (designated Le-R) rats. Disease susceptibility in randomly bred animals appeared to increase with increasing age. In the small group of young Le-R rats, which were susceptible, disease onset was delayed, severity of symptoms was reduced, and duration of clinical signs was abbreviated compared to conventional Lewis rats. The severity of histologic neural tissue lesions correlated with clinical observations. Breeding experiments indicated that most Le-R rats were resistant to disease induction regardless of whether their ancestors had been selected for susceptibility or resistance. The F3 generation of resistant lineage was uniformly resistant at all ages tested. Virtually all (Lewis X Le-R)F1 rats of either sex were resistant when challenged at 7-8 wk of age indicating that resistance was a dominant autosomal trait. Approximately half of (F1 X Lewis) backcross rats developed paralytic EAE whereas one-fourth were entirely resistant, suggesting that disease resistance may be mediated by one or two genes. Le-R rats shared at least some of the Lewis rat major histocompatibility antigens. Resistance apparently did not reflect a nonspecific impairment of cellular immune responsiveness. Le-R rats, which had been challenged with myelin basic protein, developed antigen-reactive cells specific for basic protein or its encephalitogenic fragment. Spleen cells obtained from basic protein-sensitized Le-R rats did not adoptively transfer disease into Lewis rats. In contrast, spleen cells obtained from basic protein-sensitized Lewis rats readily transferred disease into both Lewis and Le-R recipients. These data suggest that disease resistance may be a result of an immunologic deficit (or suppressor cell activity) expressed during the differentiation of antigen-reactive cells into disease-inducing effector cells.

Progressive myelopathy in older German shepherd dogs. II. Presence of circulating suppressor cells.

Progressive myelopathy in the German shepherd dog is a degenerative neurologic disease of unknown etiology. Results presented in a previous study indicated a depression in the response to thymus-dependent mitogens by peripheral blood leukocytes obtained from dogs with progressive myelopathy. Data presented here indicate that this depressed response to mitogens was associated with the presence of peripheral blood suppressor cells. Suppressor cell activity was detected in dogs that were severely affected with PM, but was not apparent in dogs that were mildly affected. Peripheral blood leukocytes obtained from dogs with progressive myelopathy suppressed the mitogenic response to autologous lymph node cells as well as allogeneic normal canine peripheral blood cells. The suppressor cells had the capacity to suppress mixed leukocyte reactions. Suppressor cell activity was radioresistant. Both nylon wool-adherent and -nonadherent peripheral blood leukocyte populations contained suppressor activity. Suppressive activity diminished after incubation of the suppressor cells with indomethacin, suggesting that suppression may be mediated by the release of prostaglandins. Although a role for peripheral blood suppressor cells in the disease process has not yet been established, it is possible that this abnormal regulatory activity reflects an attempt by the host to control an autoimmune event.