Formation Of Soluble Immune Complexes Research Articles

Delay of LPS-induced acute lung injury resolution by soluble immune complexes is neutrophil dependent

Delay of LPS-induced acute lung injury resolution by soluble immune complexes is neutrophil dependent.Shock 32(3):276-285, 2008. To the Editor: We read with great interest the article by Wu et al. (1) describing the role of immune complexes and neutrophils in a rat model of LPS-induced lung injury. The authors found that inhibition of Fc receptors (FcRs) attenuates lung injury in LPS-treated rats supposedly due to formation of soluble immune complexes after administration of LPS. They also studied expression of FcRs receptors on purified neutrophils from rats and further showed that these receptors regulate neutrophil functions, including apoptosis. In addition, the inhibition of apoptosis of neutrophils was mediated by PI3-K and ERK pathways. We had similarly discovered that mice deficient in FcRs are protected from LPS-induced lung inflammation/injury (2). This was most likely due to inability of immune complexes such as anti-KC[chemokine(CXC motif) ligand 1 (CXCL1)] autoantibody:KC complexes present in these mice to interact with their specific receptors. Moreover, we showed that immune complexes (containing anti-IL-8 autoantibody:IL-8 complexes) purified from lung edema fluids from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) inhibit apoptosis of human blood neutrophils acting via FcRs (3). In addition, we observed that key components of the FcR signaling pathway, Src, Syk, PI3-K, and ERK, may be involved in regulation of neutrophil apoptosis by the complexes (3). The 2 latter proteins were also shown to control the effect of immune complexes on apoptosis of rat neutrophils by Wu et al. (1). Finally, our laboratory reported that immune complexes present in alveolar spaces of patients with ARDS may facilitate neutrophil adhesion by enhancing the level of the expression of ICAM-1 on endothelial cells in the lung (4). Moreover, Wu et al. (1) pointed out that the composition of immune complexes that appear in LPS-treated rats is not known at present. However, we have presented solid evidence that immune complexes containing autoantibodies against chemokines, IL-8 in humans, and KC in mice may contribute to the inflammatory processes in ALI/ARDS (2-5). Importantly, the levels of anti-IL-8 autoantibody:IL-8 complexes correlated with mortality in patients with well-established ARDS and were predictive of development of ARDS in patients at risk for this disease (5). Finally, the use by Wu et al. (1) of anti-human antibodies to block and study function of FcRs in rats seems controversial taking into consideration the differences in receptor types expressed by human and rat neutrophils (6). In summary, the studies by Wu et al. (1) and our group (2-5) indicate that immune complexes and FcRs may play an important role in the pathogenesis of ALI/ARDS. Anna K. Kurdowska Agnieszka Krupa Rafal Fudala University of Texas Health Science Center Tyler, Texas

Factors affecting antigen uptake by human intestinal epithelial cell lines.

We assessed the role of size, solubility, and prophagocytic cytokines interferon-gamma (IFN-gamma), and granulocyte-macrophage colony stimulatory factor (GM-CSF) in antigen uptake and kinetics by intestinal epithelial cells using keyhole limpet hemocyanin and ovalbumin. Both fluoresceinated keyhole limpet hemocyanin (3000-7500 kDa) and fluoresceinated ovalbumin (45 kDa) were internalized by human colonic epithelial cell lines, with kinetics similar to those of fluoresceinated tetanus toxoid, and there was decreased uptake of insoluble immune complexes and no enhancement in the uptake of soluble immune complexes. In addition, neither IFN-gamma nor GM-CSF altered the kinetics of uptake nor enhanced antigen internalization by the intestinal epithelial cell lines. These data suggest that regardless of the size of the soluble antigen, the presence of prophagocytic cytokines, or the formation of soluble immune complexes, fluid phase endocytosis of antigen by intestinal epithelial cells appears to be a relatively stable process.

Monitoring the formation of soluble immune complexes composed of idiotype and anti-idiotype antibodies by electron microscopy

We have previously used immunoelectron microscopy (IEM) to generate a three-dimensional map of idiotypic (Id) and isotypic epitopes on the Fab arms of HGAC 39 (Roux et al., 1987, Proc. natn. Acad. Sci. U.S.A. 84, 4984–4988), a mouse IgG3 monoclonal antibody (Mab). In this report, we analyse the geometry of the various types of immune complexes formed by the interaction of HGAC 39 with Mab directed against four mapped epitopes. Moreover, by sampling of reaction mixtures over time, we show that the kinetics of each of the subpopulations of immune complexes, as defined by geometric configuration, can be determined. The data show that for each antibody (Ab)-HGAC 39 combination the rate of immune complex formation was greatest during the first 1.5–3.5 min but that additional complexes formed through the remainder of the half hour assay period. As anticipated, complexes composed of even number units predominated (primarily dimers and tetramers) and most of these were in the form of closed rings. The data also suggest that the location and orientation of the epitopes on HGAC 39 to which the monoclonal antibodies were bound has an influence on the types of immune complexes generated. Specifically we observed that those anti-idiotype Abs that bind to the distal tip of Fab arms (i.e. in the CDR) are less likely to produce bivalently associated ringed dimers than antibodies that bind to epitopes that are proximal to the CDR and that project laterally from the surface of the Fab arms. These data are interpreted in terms of restrictions on hinge mediated flexibility and steric inhibition between adjacent Fab arms on HGAC 39.

In vitro activity of immunoconjugates between cisplatin and an anti-CA125 monoclonal antibody on ovarian cancer cell lines.

Cis-diammine dichloro platinum (II) (CDDP), is a highly potent antineoplastic agent that is used in the treatment of ovarian cancer. However, the clinical use of CDDP is restricted by its severe side effects. In order to reduce these side effects and to enhance its therapeutic efficacy, we developed specific immunoconjugates consisting of the murine monoclonal antibody OC125 and CDDP, using diethylene triamine pentaacetic acid (DTPA) as a linker. The coupling efficiencies of the different preparations synthesized, varied between 1.10 +/- 0.42 and 2.65 +/- 1.60 mol of CDDP per mol of antibody protein. Despite the chemical modification of the antibody molecule, specific binding activity of the OC125-CDDP conjugates toward the CA125 antigen was maintained as was demonstrated by means of immunohisto-/cytochemical staining of frozen sections of ovarian cancer tissue, amniotic epithelium, and the CA125 positive ovarian cancer cell line NIH:OVCAR 3. The antiproliferative activity of the immunoconjugates was tested against the human ovarian cancer cell lines NIH:OVCAR 3 and SKOV 3, applying a kinetic crystal violet microassay. Despite the promising results obtained with the specific immunostaining of the target cells, no significant antiproliferative activity of our immunoconjugates against the cell lines tested was observed. One possible explanation for the lack of antitumor activity could be the fact that CA125 is released in large amounts by the NIH:OVCAR 3 cells. This may have prevented an efficient immunotargeting of the cancer cells by the formation of soluble immune complexes.

A calcium-dependent cryoglobulin IgM kappa/polyclonal IgG.

The mechanisms responsible for cold-induced precipitation of mixed cryoglobulins are not well understood. A mixed cryoglobulin IgM kappa/IgG (type II) of a patient with Sjögren's syndrome was studied because of its unique properties. This cryoglobulin precipitated in serum but not in serum containing 10 mM EDTA. The cryoprecipitation was shown to require calcium (Ca) and was optimal at 1 mM of free Ca. Cryoprecipitation was also induced by Ba, Mn, and Sr, but not by Mg and Co. Purified IgM kappa/IgG complexes precipitated in the presence of Ca, but not IgM kappa alone. There was no significant binding of 45Ca to the purified IgM kappa, IgM kappa/IgG complexes formed with purified components, and the cryoprecipitate. The relative affinity of the radiolabeled [125I]IgM kappa for IgG was 3.6 x 10(3) liters/mol at 37 degrees C as assessed by sucrose density gradient ultracentrifugation, and increased to 1.7 x 10(4) liters/mol at 4 degrees C. The addition of Ca produced no change in the affinity at 37 degrees C and 4 degrees C. The absence of a direct effect of Ca on the Ag/antibody reaction was confirmed in experiments using polyethylene glycol as precipitating agent. In conclusion, two independent steps were responsible for the precipitation of this cryoglobulin. The first step was an efficient formation of soluble immune complexes as produced by a drop in temperature. The second step was caused by a change in the physicochemical conditions--the presence of Ca--which induced polymerization of the IgM kappa/IgG complexes.

Formation of soluble immune complexes by complement in sera of patients with various hypocomplementemic states. Difference between inhibition of immune precipitation and solubilization.

To examine whether the ability of complement to form soluble immune complexes plays a role in preventing immune complex-mediated diseases, we analyzed the capacity of complement to inhibit immune precipitation (IIP) and to solubilize preformed immune aggregates (SOL) in 23 sera of patients with various hypocomplementemic states, and we correlated the results of these studies with the clinical syndromes found in the various patients. In sera with deficiency or depletion of early classical pathway components, IIP was profoundly altered, whereas SOL was delayed but in the normal range. In contrast, in sera with C3 depletion but intact classical pathway and in properdin-deficient serum, IIP was initially preserved, whereas SOL was abolished. Since the incidence of immune complex diseases in various hypocomplementemic states correlates with the severity of IIP defects, but not with reduced SOL, it is suggested that IIP is an essential biological function of complement that prevents the rapid formation of insoluble immune complexes in vivo.

Hydralazine and isoniazid reduce the formation of soluble immune complexes by complement

Hydralazine and isoniazid reduce the covalent binding capacity of C4. Since these two drugs are known to induce a systemic lupus erythematosus (SLE)-like syndrome, it has been suggested that this reduced binding could lead to the abnormal processing of immune complexes in vivo. Complement mediated solubilization and inhibition of immune precipitation were tested in vitro in normal human serum exposed to various concentrations of the two drugs. The formation of soluble immune complexes in the assays was reduced. The concentrations needed to induce a significant decrease were (a) for solubilization: 50 mM hydralazine and 25 mM isoniazid, and (b) for inhibition of immune precipitation: 50 mM of both. Such concentrations are unlikely to be present in vivo, so that the induction of SLE by these two drugs cannot be explained exclusively by reduced formation of soluble complexes by complement.

Immune labeling of the D and E regions of human fibrinogen by electron microscopy.

Human fibrinogen was digested with trypsin to yield core fragments D and E, and antibodies were made against the isolated fragments. The Fab' fragments derived from these antibodies were mixed with native fibrinogen, resulting in the formation of soluble immune complexes. These were rotary shadowed with platinum or negatively contrasted with uranyl acetate and examined by electron microscopy. Fab' from anti-D immunoglobulin was found to be attached to the outer nodules of fibrinogen with a frequency of 79% prior to affinity purification and 91% afterward. Fab' from anti-E immunoglobulin attached to the central nodule with a frequency of 78% prior to affinity purification and 82% afterward. The evidence clearly identifies fragment D produced by plasmin or trypsin digestion of fibrinogen with the outer nodules and the single fragment E, with the central nodule.

The Role of Antibodies in the Rejection and Enhancement of Organ Allografts

This chapter examines the evidence for immunoglobulin participation in the host response to organ allografts. Alloantibodies may have in vivo effects that are both damaging and protective to grafted tissue; furthermore, the presence in vivo of alloantigens from the graft makes possible the formation of soluble immune complexes that may impair cellular effector mechanisms in addition to having phlogogenic properties. The chapter discusses the destructive effects of antibody in allotransplantation and presents evidence pertaining to the involvement of antibody in the rejection reaction. In addition, the protective effects of antibody in allotransplantation are also discussed. The fate of a vascularized organ graft can be closely dependent upon humoral factors in constant and intimate contact with graft endothelium. If the immunoglobulin is complexed to soluble antigens from the graft, deposition of immune complexes in vessels and capillaries may produce injury, or similar complexes may serve to inhibit cell–mediated effector processes. It is also possible for free immunoglobulin to cover antigenic sites and to block target cell injury by effector cells. Furthermore, the normal physiology of immunoglobulin-cell interactions and immunoglobulin-mediator activations provides a variety of potential mechanisms for qualitative and quantitative alterations in induction of the immune response and in the efficiency of effector mechanisms at a later stage.