Removal Of Immune Complexes Research Articles

Clearance of cryoglobulins in man

From the foregoing it is evident that the defective clearance of immune complexes may contribute to tissue damage seen in patients with cryoglobulins. Hypocomplementaemia, decreased erythrocyte CR1, and the nature of the immune complexes may all contribute to decreased binding of immune complexes to erythrocytes with the potential consequence of increased availability for deposition and decreased processing of immune complexes. In addition, in type II cryoglobulinaemia the nature of the immune complex namely IgM RF/IgG complexes, has been shown in some circumstances to fix C3 and C4 inefficiently in spite of detectable fluid phase complement activation. The poor C3 fixation results not only in decreased transport by erythrocytes to the RES but also in inefficient removal of immune complexes by phagocytic cells, since the Fc receptor on these cells would be acting alone without the synergy of occupied complement receptors. Persistence of immune complexes in tissues in these circumstances has two potential consequences. First, the multivalency of IgM may contribute to enlargement of these immune complexes in situ by successive trapping of antigen and antibody complexes; this process may be aided by local factors such as low temperature at the peripheries and increased protein concentration in glomerular capillary loops, which favour "cryo" precipitation. The higher avidity of RF for IgG that is surface bound rather than monomeric would also favor immune complex formation in these circumstances. Second, although there is no opsonization of the immune complex complement activation results in bystander fixation of C4 and C3 to the surrounding tissues resulting in tissue injury.

The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans. Complement- and erythrocyte complement receptor 1-dependent mechanisms.

The role of complement and its receptor on erythrocytes (CR1) in the physiologic elimination of large immune complexes from the circulation of humans was assessed. Large radiolabeled soluble tetanus toxoid- anti-tetanus toxoid complexes were injected i.v. into three normal individuals and three patients with SLE. These complexes were prepared in antibody excess and were 45S in size, fixed C and bound to E CR1 in vitro. The percentage of complexes bound in vitro was directly proportional to CR1 number/E in four normal subjects and three SLE patients. After i.v. injection into normal subjects, complexes were cleared rapidly, with a monoexponential rate constant (10.3 to 11% complexes cleared/min). In the SLE patients, clearance was best explained by two phases: the first occurred within the first minute indicating immediate trapping of a fraction of the complexes (19.5 to 25.3% of injected complexes trapped), the second was monoexponential and was similar to the normal range. A large fraction of complexes bound within the first minute to E in vivo; the percentage of binding was variable, ranging from 16.3% to 71.5% and was related to E CR1 number. In a second study complexes were injected that had been attached to autologous E by opsonization with C in vitro. Their elimination was similarly monoexponential, except in one SLE patient in whom there was significant initial trapping (30.9%). A fraction of these complexes were released from E within the first minute, the percentage release being greatest in the patient with the lowest CR1 number (81.4%). E bearing immune complexes remained in the circulation and were not transiently sequestered in the liver or spleen. This is the first study of the clearance of soluble immune complexes in vivo in humans and shows that C and CR1 on E participate in immune complex clearance reactions, and that abnormal clearance can be detected in the form of rapid removal of immune complexes from the circulation.

Blockade of clearance of immune complexes by an anti-Fc gamma receptor monoclonal antibody.

Clearance of immune complexes by the mononuclear phagocyte system is important for maintaining normal host defenses against bacterial and viral assault (1), but also contributes to the pathogenesis of a variety of immune- mediated diseases . For example, removal from the circulation of IgG-coated erythrocytes and platelets by the MPS is the sine qua non of immune-mediated cytopenias (2, 3). On the other hand, abnormally decreased removal by the MPS of smaller, soluble immune complexes may play a role in the pathogenesis of immune complex-mediated tissue damage found in such autoimmune diseases as SLE (4). Although the physicochemical nature and the size of immune complexes can influence rates of clearance and sites of deposition (reviewed in 5), interactions between immune complexes and the MPS in vivo are poorly understood. The inability to directly measure binding or internalization of immune complexes by cells in the liver and spleen has made the analysis of the molecular basis of immune complex clearance very difficult . Receptors for the Fc portion of IgG (FcgammaR) and for complement (CR) undoubtedly play a role in the removal of immune complexes, but the relative importance of these receptors is not known.

Specific hemoperfusion through agarose acrobeads.

Agarose acrobeads were produced by encapsulating polyacrolein microspheres (acrobeads) of 0.2 micron average diameter within an agarose matrix. Crosslinked agarose acrobeads of diameters ranging from 0.5 to 0.8 mm were found to be optimal spheres for specific hemoperfusion purposes. Agarose provides the biocompatibility and mechanical strength of the agarose acrobeads. Acrobeads contain a high aldehyde-group content through which various amino ligands, i.e., proteins, antigens, antibodies, enzymes, and so on, can be covalently bound in a single step under physiological pH (or other pH). Thus, antibodies, antigens, or toxic materials may be directly removed from whole blood by hemoperfusion. During in vitro and in vivo hemoperfusion trials, the content of erythrocytes, leukocytes, and thrombocytes was essentially unaltered. Likewise, a battery of the soluble blood components (Cl-, K+, Na+, Ca2+, PO3/4-), total proteins, albumin, and C'4 component of the complement cascade, as well as the enzymes SGOT, LDH, and alkaline phosphatase, remained constant within narrow limits during the hemoperfusion procedure. The chemical and physical structure of the beads is stable; neither acrolein nor bead fragments were detected in hemoperfusion trials. Similarly, leakage of antibody bound to the agarose acrobeads into the blood is insignificant. Thus far, we have demonstrated the efficacy of the crosslinked agarose acrobeads for extracorporeal removal of "unwanted" substances from whole blood in the following systems: (a) removal of specific antigens (digoxin or paraquat removal with antidigoxin or antiparaquat antibodies bound to the acrobeads, respectively), (b) removal of specific antibody (antiBSA) removal with BSA bound to the beads), (c) removal of immune complexes (BSA-antiBSA complex removal with C1q bound to acrobeads), and (d) removal of specific metals (removal of iron with deferoxamine bound to the agarose acrobeads).

In vitro Induction of Suppressor Cells by Plasma of Rats with Trypanosoma brucei rhodesiense Infection

Mitogenic responses of B and T lymphocytes from spleens of rats infected with Trypanosoma brucei rhodesiense were suppressed. Plasma from infected rats suppressed the mitogenic responses of B and T lymphocytes from spleens of normal uninfected rats. Removal of immune complexes from plasma of infected rats significantly reduced the suppressive effect of the plasma on splenic lymphocytes of normal uninfected rats. Normal thymus cells treated with plasma from infected rats and added to cultures of normal spleen lymphocytes inhibited the mitogenic responses of B and T lymphocytes. We suggest that the interaction of immune complexes and Fc or C3b receptors of T lymphocytes resulted in the in vitro induction or activation of T suppressor lymphocytes.

Acquired immunodeficiency syndrome (AIDS)--apheresis and operative risks.

The removal of immune complexes, autoantibodies and suppressor factors from the circulation of patients with AIDS and AIDS related conditions by plasmapheresis and selective immunoadsorption may play a role as a therapeutic modality in these disorders. Lymphocytapheresis may also be of potential use in AIDS related conditions with presumed autoimmune basis. Perfusion of plasma over immobilized protein A columns is being evaluated as a possible immunomodulatory and antitumor therapy in patients with AIDS related Kaposi's sarcoma. Although apheresis procedures as a therapeutic modality in AIDS related conditions are still at the experimental stage, preliminary results are encouraging. The possible transmission of the disease by blood products presents a health hazard to health workers involved in the field of apheresis. Since the mode of transmission of the disease appears to be similar to hepatitis B, strict hepatitis B precautions should be enforced in every case in which AIDS suspected blood is being processed.

Desorption of immunoglobulins from Protein A-Sepharose CL-4B under mild conditions

Conditions which permit the dissociation of IgG-staphylococcal protein A interactions with resort to low pH buffers or the use of chaotropic ions are discussed in relation to: (a) the mechanism of the binding reaction; (b) the use of immobilised protein A for purification of cells or removal of immune complexes from serum. Glycyl-tyrosine, glycyl-histidine, glycyl-phenylalanine and tryptophan, representing the class of competing ligand desorbents, and ethylene glycol, an agent which disrupts hydrophobic interactions, were found to be the best desorbents.

Therapeutic trial of cryofiltration in patients with rheumatoid arthritis

Cryofiltration, a new technique for on-line plasma separation and its treatment by cold filtration, enables the selective removal of immune complexes and eliminates the need for replacement proteins. Fifteen patients with rheumatoid arthritis were treated for nine to 10 consecutive sessions over a three- to five-week period. Circulating immune complexes decreased by an average of 78 percent and rheumatoid factor by 32 percent. This was accompanied by significant clinical improvement in morning stiffness, articular index, 50-foot walking time, grip strength, and target joint circumference. Cryofiltration might thus be beneficial for a subgroup of rheumatoid arthritis patients in whom conventional therapy has failed.

Antibodies to collagen in scleroderma.

Using the enzyme-linked immunosorbent assay (ELISA), we detected antibodies to interstitial (type I) and basement membrane (type IV) collagens in the sera of patients with scleroderma (systemic sclerosis). Antibodies against type IV collagen were found in significant levels in these patients and correlated with the presence of abnormal pulmonary diffusion capacity. Levels of antibodies to type I collagens also correlated significantly with pulmonary diffusion capacity. Absorption of sera with type I or type IV collagens before analysis in the ELISA eliminated reactivity in an antigen-specific pattern, indicating that these antibodies reacted with determinants specific for either type I or type IV collagens. The removal of immune complexes by ultracentrifugation had no effect on serum antibody levels. Autoantibodies to basement membrane and interstitial collagens may participate in the pathogenesis of scleroderma.

Kontinuierliche Kryofiltration von Plasma bei rheumatoider Arthritis*

Plasma exchange using either cell centrifuge or membrane filtration has been advocated for the treatment of autoimmune disorders. Major limitations to its chronic and widespread application are the requirement for plasma infusion products, loss of essential plasma substances, possible contamination of the infusion solutions and limited solute removal related to the volume of exchange. To overcome these problems a continuous cryofiltration technique has been developed for application in patients with immune complex mediated disease. Plasma containing the macromolecules to be removed is filtered from blood by an initial filter, then cooled and filtered by a second membrane to remove coldprecipitable proteins and large molecules. The treated plasma is then returned to the patient on-line. The removal of immune complex containing cryoglobulins by this technique has effected great improvement in a patient with severe unremitting rheumatoid arthritis.

Immunoadsorbent: A Study on the Selective Removal of Immune Complexes in SLE

Immunoadsorbent: A Study on the Selective Removal of Immune Complexes in SLE

Rheumatoid Vasculitis

Today, we cannot conclude comments on the treatment of immune-complex disease without paying our respects to therapeutic plasmapheresis and leukapheresis. Numerous centers are beginning to examine the value of removing either plasma or lymphocytes from patients with many rheumatic diseases. Efficacy appears to be established in hyperviscosity diseases (Waldenstrom's), myasthenia gravis, Goodpasture's syndrome, RH sensitized pregnant mothers, and thrombotic thrombocytopenic purpura. In active rheumatoid arthritis, it is a useful short-term tool but hardly cost effective. Although it would appear to be an advance from the medieval concept of purging out the "evil humors," pheresis for rheumatoid vasculitis must be considered a limited research tool, which may teach us something about mechanism of action. The mechanical removal of immune complexes by pheresis probably requires additional remittive or immunosuppressive therapy to prevent rebound in immune complex production. Effort now need to be directed to the agents that initiate production of immune complexes. Once the host's antibody system is so stimulated, we often find ourselves trying to close the barn door after the horse is long gone.

Gamma-Interferon induced by S. aureus protein A augments natural killing and ADCC.

Staphylococcus aureus produces a protein identified as protein A (SpA) with a molecular weight of 41,000 (ref. 1) which binds to the Fc region of many types of mammalian IgG2, activates complement, blocks opsonins and is both chemotactic and mitogenic. SpA has been used for various immunological purposes including removal of immune complexes, arming effector cells with antibody, distinguishing between antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killing (NK), and exerting anti-tumor activity. We have now demonstrated that SpA induces the production of gamma interferon (IFN-gamma) in nonadherent, T lymphocyte-depleted (E-), Fc receptor-bearing (FcR+) human peripheral blood lymphocytes. Our data also suggest that IFN-gamma is a potent stimulator of both NK and ADCC activity.

934 INTERACTION OP POLYMORPHONUCLEAR LEUKOCYTES (PMN) AND RESPIRATORY SYNCYTIAL VIRUS (RSV): POSSIBLE ROLE IN PATHOGENESIS OF BRONCHIOLITIS

The effect of live RSV, RSV antigen-antibody complexes, and antibody to RSV on oxidative functions of human PMN was determined by employing luminol-dependent-chemiluminescence (LDCL). The presence of immune complexes was established by Raji cell radioimmune assay (RIA) and by the presence of immune aggregates on electron microscopy. The fresh PMN were obtained from healthy adult volunteers. RSV or antibody to RSV alone failed to induce significant degree of LDCL. However, RSV and RSV antibody mixtures generated significant degree of LDCL (P<0.001). The degree of LDCL activity induced by the virus-antibody mixtures was directly proportional to the presence and concentration of RSV-specific immune complexes as detected by RIA and EM. Removal of immune complexes by ultracentrifugation effectively eliminated the LDCL responses. LDCL activity was reduced four-fold after complement depletion. These data suggest a marked activation of oxidative metabolic pathway of PMN by RSV specific immune complexes. It is proposed that PMN mobilized to respiratory epithelium during the course of RSV infection, rather than the respiratory epithelium itself may serve as the targets for immune complex deposition. Such interaction may mediate the pathogenesis of RSV infection and bronchospasm via the possible release of metabolic products such as prostaglandins or thromboxanes from activated PMN.

Defective Fc-Receptor Functions Associated with the HLA-B8/DRw3 Haplotype

Dermatitis herpetiformis is associated with the HLA-B8/DRw3 haplotype in over 90 per cent of patients, and various percentages have been reported to have circulating immune complexes. Since removal of immune complexes from the circulation is thought to depend on the Fc-receptor function of tissue macrophages, we studied this function by measuring the clearance of IgG-sensitized autologous erythrocytes in 16 patients with dermatitis herpetiformis, in normal controls with the HLA-B8/DRw3 haplotype, and in randomly selected controls. All patients were HLA-B8 positive, and all of 12 patients tested were HLA-DRw3 positive. Erythrocyte clearance was reduced in eight of the 16 patients, but did not correlate with immune-complex levels. Four of eight controls with HLA-B8/DRw3 also had delayed Fc-receptor-mediated clearance as compared with normal controls. In addition, both patients and HLA-B8/DRw3-positive controls had decreased percentages and total numbers of T cells bearing Fc receptors for IgG. These findings indicate a functional Fc-receptor defect associated with the HLA-B8/DRw3 antigens.

A controlled study of plasma exchange in the treatment of severe rheumatoid arthritis.

To learn whether the removal of immune complexes from the circulation by plasma exchange could effect an improvement in disease activity in rheumatoid arthritis (RA) patients, we performed a controlled study of 20 patients, we performed a controlled study of 20 patients with severe progressive disease which had not responded to previous therapy. Ten patients (Group 1) were hospitalized, continued on their regular anti-inflammatory medication, and given a graded course of physiotherapy. A further 10 patients (Group 2) received the same treatment as the first group with the addition of a concurrent course of plasmapheresis. Clinical measurement of disease activity after treatment revealed little difference between the two groups with a statistically significant improvement in four measures in Group 1 and in five in Group 2. Laboratory studies suggested that the intensity of plasma exchange was sufficient to remove circulating immune complexes in these patients. Our results confirm that hospitalization in itself is of benefit in the treatment of acute exacerbations of rheumatoid arthritis. The marginal improvement achieved by the addition of plasma exchange in the management of these patients (despite the removal of circulating immune complexes) makes its short-term use of questionable value in the treatment of severe rheumatoid arthritis.

Extracorporeal removal of immune complexes: Preparation and characterization of immobilized bovine conglutinin

The binding of immune complexes to immobilized bovine conglutinin (K) was studied in order to develop an ex vivo immunoadsorbent system for the removal of circulating immune complexes. K was immobilized to the surface of solid, polymeric beads of two different sizes (1–3 um and 38–63 um diameter) which were activated with N-hydroxysuccinimide ester groups. The binding of both a model immune complex, aggregated human globulin (AHG) and an actual immune complex, bovine serum albumin (BSA): anti-BSA, to the immobilized K were determined utilizing both batch and flow conditions. The binding of AHG occurred rapidly (within 15 min.) at 37°C and additional binding was attained by using a subsequent low temperature incubation (37°C, 30 min., followed by 4°C, 18 hrs.). Uptake of AHG from solution was 2.6 g/g immobilized K. The binding of the 125I-AHG to immobilized K was found to be linear with concentration over the range of 20-2,000 ug AHG/ml tested. Similarly, the binding of BSA:anti-BSA complexes in solution to immobilized K was 0.16 ug 125I-BSA:anti-BSA/ug immobilized K. Significant amounts of the bound AHG were found to be released nonspecifically in the presence of buffered protein solutions, including human serum albumin (HSA) and BSA, while bound BSA:anti-BSA complexes were not released by similar treatments. Effluents of solution perfused over immobilized K showed no toxicity upon intravenous infusion into mice. These studies suggest that K may be immobilized on a biocompatible solid support and in this state retains functional capacity to extract immune complexes from solution. Hence, this may represent a promising system for use as an extracorporeal immunoadsorbent to remove circulating immune complexes in vivo.

Role of marrow-derived monocytes and mesangial cells in removal of immune complexes from renal glomeruli.

Phagocytosis of intravenously administered immune complexes by cells in the mesangium was investigated. The model used was that of exchange marrow transplantation between Chediak-Higashi (CH) mice and syngeneic partners after X-irradiation. This model was chosen since marrow-derived macrophages could be differentiated from resident mesangial cells by the presence of the characteristic giant lysosomes in phagocytic cells of the CH mice. Injected immune complexes were cleared normally and localized in the glomerular mesangium in CH or C57BL/6J mice receiving either C57BL/6J or CH marrow. C57BL/6J mice with CH marrow injected with immune complexes prepared with reduced and alkylated antibodies accumulated many cells within the mesangium that contained both giant lysosomes and electron dense deposits. Deposits were not found in cells with subplasmalemmal microfilaments and perpheral dense bodies. Conversely, the cells in the mesangium of CH mice with C57BL/6J marrow that contained electron dense deposits were devoid of giant lysosomes. Based on these observations, we concluded that (a) marrow-derived monocytes contribute to mesangial hypercellularity after deposition of immune complexes and (b) phagocytosis of immune complexes localized in the glomerular mesangium was by marrow-derived monocytes rather than by mesangial cells.

Clinical immunologic studies in systemic lupus erythematosus

This review of recent and new directions in clinical immunologic studies of systemic lupus erythematosus (SLE) is restricted to the areas of lymphocyte surface markers, antigen binding lymphocytes, immune complexes, and lymphocyte hyporesponsiveness in lupus patients. First, it is not clear whether the T-lymphopenia observed in SLE is related to viral destruction of T cells, anti-lymphocyte antibodies, or tissue sequestration. Second, the increase in DNA-binding B lymphocytes observed in active lupus patients may be related to minor alterations in the balance of immunoregulatory T cells or to a bypass of DNA-specific helper T cells. Third, it is speculated that the removal of immune complexes which play a role in lupus glomerulitis by various extracorporeal immune absorbents may be important in the future therapy of SLE. Fourth, the mechanisms of T-lymphocyte hypofunction are unexplained. It is postulated from studies done in other diseases that this hypoactivity may be mediated by the secretion of prostaglandin or other humoral agents from one leukocyte subpopulation suppressing another potentially responsive lymphocyte subpopulation. Also an investigation into the lymphocyte subpopulation reactive with virus-infected fibroblasts may be useful in delineating immunoregulatory lymphocytes important in the pathogenesis of SLE.

Studies on antigen-antibody complexes. I. Elimination of soluble complexes from rabbit circulation.

Solid phase immunoadsorbents were prepared by coupling antigens to agarose. With this technique specific antibodies were easily isolated in large amounts. The gammaG-globulin class of antibodies isolated in this manner were not denatured as judged by their normal biological half-life in rabbits. Soluble immune complexes at fivefold antigen excess were prepared from isolated specific antibodies and HSA, human lambda-chains, human lambdaG-globulins, and a Waldenström's macroglobulin as antigens. In all these preparations a characteristic immune complex was encountered that represented the smallest stable antigen-antibody union. In the HSA-anti-HSA system they were found to be AgAb(2) complexes, and Ag(2)Ab complexes in the gammaG-anti-gammaG system. These stable complexes fixed complement ineffectively. Also, a spectrum of larger complexes was present in each system, and these complexes fixed complement effectively. With intact antibodies the disappearance curves of immune complexes from the circulation were composed of three exponential components. The immune complexes larger than AgAb(2) were quickly removed from the circulation with half-lives of 0.09-0.37 hr. Their clearance was not dependent on complement components, in that depletion of complement by cobra venom factor and aggregated gammaG-globulin did not alter the pattern of their removal from the circulation. However, when the interchain disulfide bonds of antibodies were reduced and alkylated, the removal of the lambda-anti-lambda, HSA-anti-HSA, and gammaG-anti-gammaG complexes was altered. In these experiments the disappearance curves were composed of two exponential components and the rapid removal of the greater than AgAb(2) complexes did not occur. The immune complexes prepared from reduced and alkylated antibodies fixed complement ineffectively. The presented data indicate that the rapid removal of circulating immune complexes, containing gammaG-globulin molecules as antibodies, depends primarily on the number of antibodies involved. Furthermore, complement fixation is not involved in the rapid removal of such complexes. Nevertheless, the rapid removal of immune complexes and their ability to fix complement have similarities for optimal function in that both processes require intact interchain disulfide bonds of antibodies and complexes that exceed the AgAb(2) combination.