Polyreactive immunoglobulins recognize hydrophobic parts of proteins

It was shown that polyreactive immunoglobulins (PRIG), which are capable to interact non-specifically with various antigens, could differ from so called natural antibodies, which have also the capacity to react non-specifically with various antigens, including self-antigens. The main differences are that in contrast to the natural antibodies, which mainly interact, probably, with hydrophilic epitopes, PRIG recognise and bond preferentially to hydrophobic epitopes. We can consider this binding as a new type of interaction between immunoglobulins and antigens.     

Mechanisms of interaction of polyreactive immunoglobulins and protein antigens

New features of interaction between polyreactive immunoglobulins (PRIG) and protein antigens were considered. It was shown that unlike specific antibodies, recognizing mainly hydrophilic epitops of proteins and interacting against them with high affinity according to the mechanisms "lock-and-key" and/or "induced fit", PRIG recognized and nonspecifically bound to hydrophobic patches of protein antigens. On this reason it is possible to prevent or markedly diminish PRIG-antigen interaction using the reagents that have high affinity to hydrophobic regions of proteins and therefore are capable to block these regions. The obtained data are in a good agreement with the former data concerning the kinetic and thermodynamics characteristics of PRIG-antigen interaction described by us earlier.     

Polyreactive immunoglobulins and natural antibodies are different substances

It was shown that the polyreactive immunoglobulins of intact animal or human sera and the natural antibodies of these sera have different properties. Polyreactive immunoglobulins interact non-specifically with various antigens and this interaction is strongly dependent on an exposure of hydrophobic sites by antigens and, probably, by polyreactive immunoglobulins. Tween 20 and low temperature can substantially suppress this reaction. Various non-related soluble antigens can inhibit the binding of PRIG to any immobilized denatured antigen with similar efficiency. In contrast, natural antibodies interact specifically with appropriate antigens and this interaction can be suppressed only by the same or serologically similar competing antigens. Intact sera contain appreciable amount of polyreactive immunoglobulins, apparently much higher concentration than the concentration of natural antibodies. Biological functions of polyreactive immunoglobulins still remain unknown.     

Dynamics of the interaction of polyreactive immunoglobulins with immobilized antigens]

The kinetic of polyreactive immunoglobulins (PRIG) and immobilized antigen interaction was examined at different temperatures. It was shown that this process can be described by the so-called "competitive" model, and the relatively simple method for the rate constant determination for this process was developed. According to the "competitive" model PRIG molecule could be either in "active" or in "inactive" state and dynamic equilibrium exist between "active" and "inactive" molecules which strongly depend on incubation temperature. Only "active" PRIG can interact with antigens, and this is the reason of strong temperature dependence of PRIG-antigen interaction. The data also show that the mechanism of PRIG-antigen interaction differ from that of antibody-antigen interaction.     

Lipid-protein interactions and conformation of proteinaceous macromolecules

Analysis of the data as to the mechanism of specific antibody transformation into polyreactive immunoglobulins (PRIG) shows that for this transformation it is necessary and sufficiently to deprive antibodies of lipids, which are in norm tightly bound to the antibodies. Removal of these lipids by any methods (by treatment of antibodies with chaotropic ions, low/high pH, reactive oxygen species and lipases) leads to the loose by antibodies of their specificity and acquiring the ability to react with various non-related antigens, i.e. to their conversion into PRIG. Mathematical modeling of the PRIG--antigen interaction and values of thermodynamic characteristics of this process shows that antigen-binding domains of PRIG are in semi-melted state, thanks to what they can fit their structure to be complementary to structurally different antigens. Thus, we conclude that lipids bound to the so-called "hydrophobic pockets" of immunoglobulins (Ig) can stabilize the conformation of Ig and increase their rigidity, and removal of these lipids induce flexibility of Ig domains, responsible for interaction with antigens. It was presumed that lipids could exert the similar function of conformation stabilization not only in the case of antibodies, but also combining with some other proteins, for example, enzymes. Their removal could lead to the changing of protein conformation and loosing its biological activity. In this case the function of lipid removing and protein inactivation could exert cellular reactive oxygen species and cellular lipases and lipoxygenases.     

Transformation of specific antibodies to polyreactive immunoglobulins and their binding to blood vessel walls

It was shown that 30-50% ethanol or 40-70% dimetilsulfoxide could efficiently induce in vitro transformation of specific monoclonal antibodies (mAbs) into non-specific polyreactive immunoglobulins (PRIG). Intravenous injection 0.4 ml of FeSO4-EDTA mixture (60 and 30 mkM respectively) could induce increase of PRIG reactivity in the blood-stream. Intramuscle injection of either 0.1 ml of 40% ethanol, or 0.1 ml of FeSO4-EDTA mixture into muscle of hind limb of C57B1 mice leads to the substantial binding of circulated immunoglobulins to the blood vessels of the muscle. The similar effect could also be induced by ischemia/reperfusion of mice hind limb. In the case of intravenous injection of specific to ovalbumin biotinilated mAbs, the subsequent intramuscle injection of 0.1 ml of 40% ethanol induces apparent transformation of these mAbs into PRIG and their binding to the blood vessels. Intramuscle injection of 0.1 ml of FeSO4-EDTA mixture induces less than ethanol though noticeable effect. The obtained data have shown that cord-blood circulating specific antibodies could be transformed into PRIG at some conditions in vivo. If so, this process might play an important role in the organism defence against infections but could, probably, facilitate the development of atherosclerosis, cardiac infarct, cerebral stroke or tumors.     

Infection, autoimmune diseases, atherosclerosis, infarct, stroke, cancerogenesis: what is in common

The problem of the transformation of specific antibodies into nonspecific polyreactive immunoglobulins (PRIG) was considered. On the basis of the obtained data the conclusion has been made that this process can occur not only in vitro, but also in vivo. The biological consequences of such a transformation of circulated specific antibodies into PRIG or increase of PRIG reactivity because of their unblocking are discussed. It was supposed that PRIG was able to play positive role in the defence of organisms against some infections. Meanwhile, disregulation of the PRIG production in vivo can, probably, lead to their participation in induction and/or in aggravation of some danger diseases, such as autoimmune diseases, atherosclerosis, infarct, stroke, and cancer. If so, farther investigation of PRIG involvement in these pathological processes could open new ways for their curing or even prophylaxis.     

Mechanisms for increasing the activity of polyreactive immunoglobulins in vivo

The mechanisms of increasing of polyreactive immunoglobulin (PRIG) activity in vivo are suggested. It was shown that at some conditions, which can be observed in organism in sites of inflammation, activity of serum PRIG could be enhance considerably. This enhancing might be induced either by reactive oxygen species, mainly by hydroxyl radicals, or by lipases. The increasing of PRIG activity could be not only a result of preexisting PRIG unblocking, but also a result of transformation specific antibodies into PRIG, caused by peroxide degradation or lipolysis of lipids, tightly but noncovalently linked to antibodies, either by hydroxyl radicals or lipase. It was also suggested that most (if not all) specific antibodies consist of not only polypeptide chains, but also some lipids. Deprived of these lipids antibodies loose their specificity and transform into polyreactive immunoglobulins.     

Study of the nature and mechanism of biosynthesis of nonspecific immunoglobulins]

Mice were immunized with Vi-antigen. Spleen cells, removed at different time after immunization, were cultivated in Eagle medium, containing glycine-14C. The biosynthesis of antibodies to Vi-antigens, autoantibodies to mouse IgG and antigen-dependent non-specific immunoglobulins (NigG) were determined by means of specific immunosorbents. Immunization of mice with Vi-antigen resulted in a sharp increase in antigen-dependent NIgG formation. Thus, this protein biosynthesis takes place not only during immunization with thymus-dependent antigens, but also in response to the thymus-independent antigen. It is shown that the synthesized antigen-dependent NIgG were not autoantibodies to self mouse IgG.     

Suppression of growth of guinea pig line-10 hepatocarcinoma. I. Effect of simultaneous administration of tumor cells and antibodies from normal rabbits.

Suppression of growth of the line-10 hepatocarcinoma in strain-2 guinea pigs occurred when line-10 cells were injected intradermally together with sera or immunoglobulins derived from normal rabbits. A significant number of animals were resistant to subsequent rechallenge with tumor cells. This immunity was specific, depended on contact of immunoglobulins with tumor cells and on the concentration of immunoglobulins. Repeated injections acted as potent vaccines and resulted in the development of immunity in 84.6% of recipients. Fc receptors were not detected on line-10 cells. Antibodies reacting with line-10 cell unique antigens as well as with antigens common to line-10, line-1 and normal guinea pig spleen cells were found in NRS. Injection of line-10 cells together with rabbit immunoglobulins from which antibodies reacting with antigens derived from line-10 cells had been removed did not result in tumor suppression. The specific antigen(s) recognized by antibodies that suppressed growth of the line-10 tumor in vivo was not determined.     

Bovine Immunoglobulin/Protein Isolate Binds Pro-Inflammatory Bacterial Compounds and Prevents Immune Activation in an Intestinal Co-Culture Model

Intestinal barrier dysfunction is associated with chronic gastrointestinal tract inflammation and diseases such as IBD and IBS. Serum-derived bovine immunoglobulin/protein isolate (SBI) is a specially formulated protein preparation (>90%) for oral administration. The composition of SBI is greater than 60% immunoglobulin including contributions from IgG, IgA, and IgM. Immunoglobulin within the lumen of the gut has been recognized to have anti-inflammatory properties and is involved in maintaining gut homeostasis. The binding of common intestinal antigens (LPS and Lipid A) and the ligand Pam3CSK4, by IgG, IgA, and IgM in SBI was shown using a modified ELISA technique. Each of these antigens stimulated IL-8 and TNF-α cytokine production by THP-1 monocytes. Immune exclusion occurred as SBI (≤50 mg/mL) bound free antigen in a dose dependent manner that inhibited cytokine production by THP-1 monocytes in response to 10 ng/mL LPS or 200 ng/mL Lipid A. Conversely, Pam3CSK4 stimulation of THP-1 monocytes was unaffected by SBI/antigen binding. A co-culture model of the intestinal epithelium consisted of a C2BBe1 monolayer separating an apical compartment from a basal compartment containing THP-1 monocytes. The C2BBe1 monolayer was permeabilized with dimethyl palmitoyl ammonio propanesulfonate (PPS) to simulate a damaged epithelial barrier. Results indicate that Pam3CSK4 was able to translocate across the PPS-damaged C2BBe1 monolayer. However, binding of Pam3CSK4 by immunoglobulins in SBI prevented Pam3CSK4 translocation across the damaged C2BBe1 barrier. These results demonstrated steric exclusion of antigen by SBI which prevented apical to basal translocation of antigen due to changes in the physical properties of Pam3CSK4, most likely as a result of immunoglobulin binding. This study demonstrates that immunoglobulins in SBI can reduce antigen-associated inflammation through immune and steric exclusion mechanisms and furthers the mechanistic understanding of how SBI might improve immune status and reduce inflammation in various intestinal disease states.     

Activation of "silent" antibodies and their interaction with antigens

Animal and human blood serum contains great amount of blocked (or "silent") immunoglobulins which, being activated by heating to 60 degrees C, pH decrease to 2.0-2.5 or treatment with 5M KSCN acquire a capacity to interact with different antigens. This interaction may be equally prevented or weakened by both identical and serologically non-related antigen, i.e. activated immunoglobulins are polyspecific. Polyspecific immunoglobulins show less affinity in comparison with monospecific antibodies, their interaction with antigens depends considerably on temperature.     

Exogenous C1q reconstitutes resident but not inflammatory mouse peritoneal macrophages for Fc receptor-dependent cellular cytotoxicity and phagocytosis. Relationship to endogenous C1q availability

Murine resident peritoneal macrophages (PM) were refractory to activation for antibody-dependent cellular cytotoxicity (ADCC) of SRBC targets as compared with either oil or thioglycollate-elicited inflammatory macrophages. Western blot analysis of macrophage cellular lysates indicated a direct correlation between the endogenous C1q levels and their innate response to activation for ADCC. Inflammatory PM had 7- to 14-fold higher C1q levels (ca. 23 to 45 ng C1q/100 micrograms protein) than resident PM (ca. 3 ng C1q/100 micrograms protein) as determined by densitometric scanning of blots. Purified exogenous mouse or human C1q were found to reconstitute the response of resident PM for ADCC mediated by C-activating mouse IgG2a or IgG2b mAb, but not by non-C-activating IgG1. Thioglycollate-elicited PM with highest endogenous C1q levels were unaffected by exogenous C1q, whereas oil-elicited PM with intermediate C1q levels were slightly augmented in their ADCC response by exogenous C1q. Augmentation of the resident PM response for ADCC activation was accomplished by either coincubation of effector macrophages with physiologic concentrations of C1q (0.5 to 4.0 micrograms/ml), IgG, and SRBC targets or by IgG and C1q preopsonized targets. FcR-dependent phagocytosis by resident PM was similarly reconstituted by exogenous C1q. The results indicate that resident macrophages with low potential for C1q biosynthesis and secretion were reconstituted by exogenous C1q in their FcR-dependent phagocytosis and ADCC, whereas inflammatory macrophages with sufficient endogenous C1q levels were largely unaffected. Thus C1q appears to have a pivotal mechanistic role in the initiation of macrophage activation for FcR-dependent effector functions.     

Exogenous C1q reconstitutes a secondary deficiency of C5-deficient AKR mouse macrophages for FcR-dependent cellular cytotoxicity and phagocytosis

Studies originally designed to assess the putative role of endogenous C5 in macrophage activation for antibody-dependent cellular cytotoxicity (ADCC) yielded unanticipated results. Resident and inflammatory peritoneal macrophages from C5-deficient AKR mice were found to have significantly lower capacity for FcR-dependent ADCC activation and phagocytosis of IgG-opsonized SRBC targets than did C5-competent C3HeB/FeJ (C3H) mice. Reconstitution of the ADCC response of AKR macrophages was accomplished initially with C5-sufficient C3H mouse serum, which suggested that endogenous C5 may be required for ADCC activation. However, further investigation largely eliminated C5 involvement in that a heat-labile component of C5-deficient AKR serum was shown to be active in the reconstitution of ADCC activation of AKR macrophages. Macrophages from AKR mice were found to have significantly lower levels of C1q mRNA synthesis, endogenous C1q levels, and C1q secretion than did C3H mouse macrophages as determined by Northern blot, Western blot, and presynthetic radiolabeling analysis, respectively. The addition of purified exogenous C1q to IgG-opsonized SRBC targets fully reconstituted ADCC activation for AKR inflammatory peritoneal macrophages to levels of normally FcR-responsive C3H macrophages. Similarly, exogenous C1q augmented FcR-dependent phagocytosis of AKR macrophages but had no effect on macrophages from responsive C3H mice. Our results indicate that AKR mice have a deficiency for FcR-dependent cellular cytotoxicity and phagocytosis that is related to their low potential for C1q synthesis and secretion rather than to their established genetic deficiency for C5 synthesis. We tentatively conclude that endogenous C1q is required as an accessory molecule for macrophage FcR-dependent effector functions and that C5 is not a prerequisite for ADCC activation.     

Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins

Complement component C1q is a protein complex of the innate immune system with well-characterized binding partners that constitutes part of the classical complement pathway. In addition, C1q was recently described in the central nervous system as having a role in synapse elimination both in the healthy brain and in neurodegenerative diseases. However, the molecular mechanism of C1q-associated synapse phagocytosis is still unclear. Here, we designed monomer and multimer protein constructs, which comprised the globular interaction recognition parts of mouse C1q (globular part of C1q [gC1q]) as single-chain molecules (sc-gC1q proteins) lacking the collagen-like effector region. These molecules, which can competitively inhibit the function of C1q, were expressed in an Escherichia coli expression system, and their structure and capabilities to bind known complement pathway activators were validated by mass spectrometry, analytical size-exclusion chromatography, analytical ultracentrifugation, CD spectroscopy, and ELISA. We further characterized the interactions between these molecules and immunoglobulins and neuronal pentraxins using surface plasmon resonance spectroscopy. We demonstrated that sc-gC1qs potently inhibited the function of C1q. Furthermore, these sc-gC1qs competed with C1q in binding to the embryonal neuronal cell membrane. We conclude that the application of sc-gC1qs can reveal neuronal localization and functions of C1q in assays in vivo and might serve as a basis for engineering inhibitors for therapeutic purposes.     

Cationization of protein antigens. II. Alteration of regulatory properties

Immunoregulatory effects of cationized bovine serum albumin (cBSA) and native bovine serum albumin (nBSA) have been investigated. Intravenous administration of nBSA to BDF1 mice substantially suppressed the antibody response to subsequent immunization with either nBSA or cBSA, whereas pretreatment with cBSA by the same route significantly enhanced the responses to both antigens. The functional properties of BSA-specific T and B cells from mice immunized with cBSA or nBSA were examined in reconstitution experiments in which splenic T populations together with B cells were transferred into irradiated syngeneic recipients. Transfer of splenic T cells from mice primed with nBSA caused profound suppression of the response to subsequent immunization with nBSA or cBSA, whereas transfer of either B or T cells from cBSA treated mice produced an enhanced response to both antigens. C57BL/6 mice, which are considered to be low responders to BSA, produced a significant antibody response to BSA when immunized with cBSA. In contrast, immunization with nBSA did not produce measureable amounts of antibody in mice of this strain. Our data clearly demonstrate that cationized BSA exhibits unique immunogenic properties due to alterations in the self-regulation of the immune response.     

Reconstitution of a deficiency of AKR mouse macrophages for their response to lipid A activation for tumor cytotoxicity by complement subcomponent C1q: role of IFN-gamma

C5-deficient AKR mouse macrophages were initially found to be refractory to activation by lipid A to mediate tumor cytotoxicity for P815 mastocytoma or L1210 mouse leukemia targets as compared with responsive C3H mouse macrophages. The lower level of tumor cytotoxicity by lipid A-activated AKR macrophages correlated with lower levels of cytotoxic nitric oxide generation as measured by nitrite end product accumulation. The refractory state of AKR macrophages was unexpectedly found to be independent of their C5 deficiency in that IFN-gamma reconstituted their response to activation by lipid A coincident with an increase in C1q mRNA synthesis. AKR macrophages were augmented in their lipid A activation by exogenous soluble C1q in the absence of IFN-gamma, which corresponded with an increased production of nitric oxide by C1q-reconstituted macrophages. In contrast, responsive C3H mouse macrophages with sufficient levels of C1q synthesis were inhibited by exogenous soluble monomeric C1q in their lipid A activation. Both AKR and C3H macrophages plated over immobilized C1q were inhibited in their lipid A activation for tumor cytotoxicity and nitric oxide generation. Our results provide evidence that C1q modulates macrophage activation by lipid A for nitric oxide-mediated tumor cytotoxicity under the influence of IFN-gamma, which stimulates C1q synthesis and secretion. These findings strongly suggest that macrophage synthesis of C1q, but not C5, is a prerequisite for their activation by lipid A.     

Structural and functional studies in C1q deficiency

The sera of two brothers were found totally lacking hemolytic C activity. One of them, a 16-yr-old male, presented a severe lupus-like syndrome, whereas the other was apparently healthy. Immunochemical quantitation of C components in both sera showed depressed levels of C1q, whereas the levels of C1r, C1s, and C1 inhibitor were elevated. C4, C3, C5, factor B, and beta 1H levels were in the normal range. Hemolytic C1 activity was totally lacking. C4 titers were elevated (150% of normal). C2 hemolytic activity was about one-third of normal, and the titers of the terminal components C3-C9 were also reduced in the two siblings. Double immunodiffusion against anti-C1q antiserum showed a partial loss of C1q antigenic determinants in the two siblings. Furthermore, the C1q of both siblings was unable to interact with immunoglobulins or to associate with C1r and C1s. Addition of purified human C1q to the sera restored their total C and C1 hemolytic activity. The dose response to the C1q addition was linear, indicating that the functional deficiency was not due to the presence of a serum inhibitor. Although antigenically deficient in comparison with normal C1q, the abnormal C1q appeared to have a larger m.w., as determined by gel chromatography. Investigation of other members of this family suggests a genetically linked disorder, because four out of six siblings had the same dysfunctional C1q in their serum.     

The extracellular matrix and inflammation: fibromodulin activates the classical pathway of complement by directly binding C1q

Components that propagate inflammation in joint disease may be derived from cartilage since the inflammation resolves after joint replacement. We found that the cartilage component fibromodulin has the ability to activate an inflammatory cascade, i.e. complement. Fibromodulin and immunoglobulins cause comparable deposition of C1q, C4b, and C3b from human serum. Using C1q and factor B-deficient sera in combination with varying contents of metal ions, we established that fibromodulin activates both the classical and the alternative pathways of complement. Further studies revealed that fibromodulin binds directly to the globular heads of C1q, leading to activation of C1. However, deposition of the membrane attack complex and C5a release were lower in the presence of fibromodulin as compared with IgG. This can be explained by the fact that fibromodulin also binds complement inhibitor factor H. Factor H and C1q bind to non-overlapping sites on fibromodulin, but none of the interactions is mediated by the negatively charged keratan sulfate substituents of fibromodulin. C1q but not factor H binds to an N-terminal fragment of fibromodulin previously implicated to be affected in cartilage stimulated with the inflammatory cytokine interleukin 1. Taken together our observations indicate fibromodulin as one factor involved in the sustained inflammation of the joint.     

Murine cutaneous leishmaniasis: resistance correlates with the capacity to generate interferon-gamma in response to Leishmania antigens in vitro

The kinetics of cell-mediated immunity developed during the course of Leishmania major infection in resistant (C57BL/6) and susceptible (BALB/c) mice were determined by using in vitro bioassays. Cells isolated from the lymph nodes draining the infected footpads were assayed for their proliferative responses to leishmania antigens (promastigote and amastigote) or to concanavalin A (Con A). Although lymph node cells (LNC) from both mouse strains proliferated to mitogen and antigen early after infection, both C57BL/6 and BALB/c mice developed diminished in vitro proliferative reactivity within 3 to 5 wk after infection. LNC from both mouse strains recovered lymphoproliferative reactivity to Con A (week 6), but only C57BL/6 mice regained reactivity to leishmania antigens. BALB/c cells remained unresponsive to leishmania antigens throughout the subsequent course of the infection. Supernatants derived from cultures of LNC that had been stimulated with Con A or leishmania antigens were assayed for interferon-gamma (IFN-gamma) by analyzing three distinct activities associated with IFN-gamma. Culture supernatants derived from leishmania antigen stimulation of LNC from infected C57BL/6 mice, but not BALB/c mice, were able to induce surface Ia on murine P388D1 cells. Ia-inducing activity was detectable in supernatants from C57BL/6 cells as early as 3 wk, and peaked by 5 wk after infection. Although cells from infected BALB/c mice never produced detectable IFN-gamma in response to leishmania antigens, LNC from both mouse strains produced readily detectable IFN-gamma in response to Con A throughout the course of infection. Culture supernatants that induced Ia on P388D1 cells were also capable of activating resident peritoneal macrophages to display leishmanicidal activity and of inhibiting encephalomyocarditis virus replication in murine fibroblasts. Each of these activities could be removed by prior incubation of the supernatants with rabbit heterologous anti-murine IFN-gamma sera or monoclonal rat-anti-murine IFN-gamma. The correlation of healer status with the capacity to generate IFN-gamma in vitro in response to leishmania antigens was examined in BALB/c mice that had been exposed to sublethal irradiation (550 rad) before infection. These animals have been previously shown to effectively resist L. major infection. Consistent with observations in the genetically resistant C57BL/6 mice, LNC from these animals demonstrated the capacity to respond to in vitro leishmania antigen stimulation with lymphoproliferation, and more importantly, by producing IFN-gamma.(ABSTRACT TRUNCATED AT 400 WORDS)