Tài liệu Báo cáo khoa học: Exposure of IgG to an acidic environment results in molecular modifications and in enhanced protective activity in sepsis doc

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Tài liệu Báo cáo khoa học: Exposure of IgG to an acidic environment results in molecular modifications and in enhanced protective activity in sepsis doc

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Exposure of IgG to an acidic environment results in molecular modifications and in enhanced protective activity in sepsis Iglika K. Djoumerska-Alexieva 1, *, Jordan D. Dimitrov 1,2,3,4, *, Elisaveta N. Voynova 1 , Sebastien Lacroix-Desmazes 2,3,4 , Srinivas V. Kaveri 2,3,4 and Tchavdar L. Vassilev 1 1 Department of Immunology, Stefan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria 2 Centre de Recherche des Cordeliers, Universite ´ Pierre et Marie Curie Paris 6, France 3 Universite ´ Paris Descartes, France 4 INSERM U 872, Eq. 16, Paris, France Introduction The ability of antibodies to interact with one single or with multiple structurally unrelated antigens (monore- activity versus polyreactivity) is believed to be an inherent property of each individual immunoglobulin molecule. However, it has been previously shown by us, as well as by others, that the in vitro exposure of monoclonal and of polyclonal IgG to various protein- destabilizing factors may result in dramatic enhance- ment of their binding polyreactivity. These treatments include high-salt solutions, low-pH or high-pH buffers, chaotropic agents, ferrous ions, reactive oxygen species, and heme [1–7]. Keywords antibodies; antibody polyreactivity; antigen– antibody interaction; IgG; immunoglobulins Correspondence T. Vassilev, Stefan Angelov Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev St., Block 26, 1113 Sofia, Bulgaria Fax: +359 2 870 0109 Tel: +359 2 979 6348 E-mail: vassilev@microbio.bas.bg *These authors contributed equally to this work (Received 22 February 2010, revised 22 April 2010, accepted 18 May 2010) doi:10.1111/j.1742-4658.2010.07714.x IgG molecules are exposed on a regular basis to acidic conditions during immunoaffinity purification procedures, as well as during the production of some therapeutic immunoglobulin preparations. This exposure is known to induce in them an antigen-binding polyreactivity. The molecular mecha- nisms and the possible biological significance of this phenomenon remain, however, poorly understood. In addition to the previously reported ability of these modified IgG antibodies to interact with a large panel of self-anti- gens, enhanced binding to non-self-antigens (bacterial), an increased ability to engage in F(ab¢) 2 ⁄ F(ab¢) 2 (idiotype ⁄ anti-idiotype) interactions and an increased functional antigen-binding affinity are reported here. The newly acquired ‘induced polyreactivity’ of low-pH buffer-exposed IgG is related to structural changes in the immunoglobulin molecules, and is at least partly attributable to the enhanced role of the hydrophobic effect in their interactions with antigen. Our results suggest that data from many previous studies on monoclonal and polyclonal IgG antibodies purified by low-pH buffer elution from protein A or protein G immunoaffinity columns should be reconsidered, as the procedure itself may have dramatically affected their antigen-binding behavior and biological activity. Low-pH buffer-trea- ted pooled therapeutic immunoglobulins acquire novel beneficial properties, as passive immunotherapy with the pH 4.0 buffer-exposed, but not with the native therapeutic intravenous immunoglobulin preparation, improves the survival of mice with bacterial lipopolysaccharide-induced septic shock. Abbreviations ANS, 8-anilinonaphthalene-1-sulfonate; CRP, C-reactive protein; IFN-c, interferon-c; IVIg, intravenous immunoglobulin; LPS, lipopolysaccharide; RU, relative units. FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3039 A comparative study of seven licensed commercially available pooled therapeutic intravenous immunoglob- ulins (IVIgs) revealed that those produced using a frac- tionation step at low pH were significantly more polyreactive when tested on a complex mix of self-anti- gens [8]. The molecular mechanisms responsible for the effect of low-pH buffer exposure on IgG molecule have remained, however, poorly understood. IVIg preparations with lower and with higher antigen-bind- ing polyreactivity have quantitatively different effects on cells in vitro. Low-pH buffer-exposed IVIg causes significantly stronger suppression of Phaseolus vulgaris agglutinin-induced proliferation of human peripheral blood mononuclear cells than the native preparation [8]. Polyreactive natural antibodies form part of the innate immunity mechanism, and are known to play a major role in preventing pathogen dissemination in the preimmune host [9–11]. Natural polyreactive antibod- ies are detected in the sera of all healthy individuals, and their immunoreactivity increases dramatically in the pure IgG fractions, purified from the same sera using low-pH buffer elution from immunoaffinity col- umns [12]. However, the physicochemical characteris- tics and the biological activities of IgG antibodies transiently exposed to low-pH conditions remain unknown. We hypothesized that low-pH (£ 4) buffer exposure could endow a commercially available IVIg preparation with novel beneficial therapeutic proper- ties. The present study shows that low-pH buffer treat- ment of a commercial IVIg results in its enhanced binding to bacterial antigens as well as to self-antigens, owing to structural changes in the immunoglobulin molecules. The modified preparation is shown to have a protective effect in experimental sepsis. Results Exposure to a low-pH buffer increases the binding polyreactivity of IgG to foreign antigens Previous studies showed that low-pH buffer-exposed IVIg acquired enhanced autoreactivity [8]. The first aim of the study was to find out whether the same broadening of IgG polyreactivity occurred when for- eign antigens were used. The exposure of pooled human IgG to a pH 4 buffer resulted in an increase in its pre-existing binding to antigens present in an Escherichia coli lysate, and in the appearance of some new bands showing the acquisition of new antibody reactivities (Fig. 1A). Interestingly, the same treatment did not significantly change the reactivity to Bacillus anthracis antigens. In contrast, the transient exposure of IVIg to pH 2.8 buffer resulted in a significant (P < 0.05) enhancement of immunoreactivity, and in A B C E D Fig. 1. Exposure of IgG to a pH 4 buffer results in increased antigen-binding polyreactivity. (A) Densitometric profiles of the reactivity of the native (solid line) and low-pH buffer-exposed (dashed line) IVIg to Escherichia coli antigens. Migration distances (x-axis) expressed in pixels were plotted against the intensity of binding (y-axis) expressed in relative units (RU) for each IVIg preparation. (B) Reactivity of native (lines 1 and 4), pH 4 buffer-exposed (lines 2 and 5) and pH 2.8 buffer-exposed (lines 3 and 6) IVIg with Bacillus anthracis antigens. The membranes were incubated with two concentrations of IVIg: 100 lgÆmL )1 (lines 1–3) and 50 lgÆmL )1 (lines 4–6). (C) Increased binding of pH 4 buffer- exposed IVIg to recombinant human IFN-c. (D) Increased binding of low-pH buffer-exposed IVIg to human factor H. In both panels, binding of the native IVIg is indicated by a solid line, and that of the low-pH buffer-exposed IVIg is indicated by a dashed line. (E) Binding of two commercially available IVIg preparations [Endobulin (solid line); Octagam (dashed line)] to human factor H. Data represent mean absorbance values ± standard deviation of quadruplicate wells in one of three ELISA experiments. Molecular modifications in low pH-exposed IgG I. K. Djoumerska-Alexieva et al. 3040 FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS the appearance of a number of novel antigen-binding specificities (Fig. 1B). Enhanced binding of low-pH buffer-exposed IVIg to recombinant human interferon-c (IFN-c) and other self-antigens Interactions of IVIg with cytokines and other mole- cules of the immune system have been shown to play a role in the immunomodulatory effect of the prepara- tion [13]. To determine whether the IgG treatment described affected binding to a typical proinflammato- ry cytokine, the interactions of the native and the low-pH buffer-exposed IVIg preparations with recom- binant human IFN-c were compared by ELISA. The pH 4 buffer-exposed IVIg showed significantly (P < 0.05) stronger binding to this human proinflam- matory cytokine (Fig. 1C). Next, the reactivity of native and of low-pH buffer (pH 2.8)-exposed IVIg towards a panel of structurally unrelated pure plasma proteins or intracellular self-proteins was analyzed. This transient exposure resulted in increases in their binding to all tested target antigens [see Fig. 1D for reactivity to factor H; data not shown for all other antigens (see Experimental procedures)]. We also com- pared the antigen-binding potentials of two commer- cially available IVIg preparations that differ in the absence or presence of exposure to low-pH conditions during the production process (Endobulin versus Octa- gam). As expected, the binding of the second to fac- tor H was significantly (P < 0.05) higher (Fig. 1E). The same was true for all other antigens in the panel (not shown). Low-pH buffer exposure increases the anti-idiotypic reactivity of IgG After a pH 4 buffer exposure, the studied IVIg prepa- ration showed enhanced binding to IVIg F(ab¢) 2 frag- ments (Fig. 2A) as well as to autologous pooled IgM molecules (Fig. 2B). In contrast, no increase in reactiv- ity of the modified IVIg to Fc-c (Fig. 2C) or Fc-l fragments (Fig. 2D) was observed. Antigen-binding kinetics of low-pH buffer-exposed IVIg In order to obtain quantitative information on the effect of low-pH buffer exposure on the reactivity of IgG, we used real-time kinetic measurements of the interaction of IVIg with C-reactive protein (CRP). The binding profiles obtained after single injections of native and of low-pH buffer-exposed IVIg were com- pared. As shown in Fig. 3A, transient (5 min) exposure of IVIg to pH 2.8 buffer resulted in an increase in the reactivity towards human CRP, as detected by this nonequilibrium binding assay. The reactivity of the pH 4 buffer-exposed preparation was also elevated, but to a much lower extent (approximately nine-fold). The native IVIg preparation showed no detectable binding to CRP at the concentration and during the period of observation used in the experiment. Interaction analyses using increasing concentrations of the native and of the low-pH buffer-treated IVIg were also performed (Fig. 3B). The data obtained were used to evaluate the kinetic constants of these inter- actions. The bimolecular association rate constant of Fig. 2. Low-pH buffer exposure of pooled human IgG enhances its binding to F(ab¢) 2 immunoglobulin fragments. Dialysis of IVIg against a pH 4 buffer results in increased binding to F(ab¢) 2 fragments of IVIg (A) and to pooled human IgM (B), but not to Fc-c (C) or Fc-l fragments (D), as assessed by ELISA. Data represent mean absorbance values ± standard deviation of quadruplicate wells (solid lines, native IVIg; dashed lines, low-pH buffer-exposed IVIg). I. K. Djoumerska-Alexieva et al. Molecular modifications in low pH-exposed IgG FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3041 binding of the pH 2.8 buffer-exposed IVIg to CRP was very low, at 33.5 ± 1.3 mol )1 Æs )1 . The estimated dissociation rate constant had a value of (2.32 · 10 )3 ) ± (2.0 · 10 )5 s )1 ). The equilibrium dissociation constant for CRP and low-pH buffer-exposed IVIg was 69.1 lm. The absence of detectable binding or negligible responses precluded the estimation of reli- able values of kinetic parameters for the native and the pH 4 buffer-exposed IVIg preparations. Enhanced role of hydrophobicity in the binding of a pH 4.0 buffer-treated monoclonal IgG antibody to its target antigen To evaluate the types of intermolecular interactions in antigen binding, pH and salt concentration screening assays were performed. This study was carried out using the mouse monoclonal Z2 antibody, which behaves in its native form as a typical monoreactive antibody, as it interacts only with mouse IgG 2a [14]. The interaction of the native Z2 antibody with its immobilized target antigen was highly pH-dependent, and characterized by a bell-shaped curve with a bind- ing optimum at neutral pH (Fig. 4A). On the other hand, the interaction of low-pH buffer-exposed Z2 antibody was much less dependent on the pH of the buffer. From pH 4.5 upwards, the binding reached a plateau and became almost independent of further increases in pH. The salt concentration dependence of the same inter- action (within the range 0–4 m sodium chloride) was also studied. The binding of the native Z2 antibody to IgG 2a was shown to be highly dependent on the salt concentration in the buffer. In contrast, this interac- tion was mostly independent of the salt concentration in the case when the low-pH buffer-exposed Z2 was left to bind with its target antigen (Fig. 4B). Both observations could be explained by an increased role for the hydrophobic effect in the interaction of the modified monoclonal antibody with its immobilized antigen. Increase in the IgG hydrophobicity as evaluated by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence In order to confirm the increased role of the hydro- phobic effect upon low-pH buffer exposure of IgG, fluorescence spectroscopy using a molecular probe for protein hydrophobicity was applied. ANS changes its A B Fig. 3. Real-time interaction analysis of the binding of IVIg to human CRP. (A) Comparison of interaction profiles of 5 lM native IVIg (black line), pH 4 buffer-exposed IVIg (gray line) and pH 2.8 buffer-exposed IVIg (light gray line) with human CRP. (B) Profiles characterizing the interactions of increasing concentrations (0.039–1.25 l M) of native IVIg (left panel), pH 4 buffer-exposed IVIg (middle panel) and pH 2.8 buf- fer-exposed IVIg (right panel) with the same human molecule. The sensorogram depicting the interaction of pH 2.8 buffer-exposed IVIg was used for evaluation of the binding affinity by global analyses. All measurements were performed at 25 °C. The results obtained in one of two independent experiments are shown. Molecular modifications in low pH-exposed IgG I. K. Djoumerska-Alexieva et al. 3042 FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS fluorescence properties with the polarity of the envi- ronment. Thus, the transition from a polar to a non- polar (hydrophobic) environment results in a dramatic increase in its fluorescence signal. This property and the ability of ANS to bind to proteins make it a widely used molecular probe for the evaluation of hydropho- bicity of proteins, as well as for exploring structural alternations in protein molecules [15–18]. Incubation of native IVIg in the presence of ANS resulted in a modest change in the fluorescence signal (Fig. 4C). Similar spectral characteristics were mea- sured in the case of IVIg exposed to a pH 4 buffer, implying the absence of a significant increase in the total hydrophobicity of the immunoglobulins. The lack of correlation between these findings and the data shown in Fig. 4A,B could well be explained by the dif- ferent IgG preparations studied (pooled, polyclonal versus monoclonal) and the different sensitivities of the methods used. Dramatic changes in the fluorescence characteristics of ANS were seen in the presence of pH 2.8 buffer-exposed IVIg. Thus, a considerable increase in the fluorescence intensity and a blue shift in the fluorescence maxima were observed. These effects were observed at different concentrations of ANS (Fig. 4D). These findings further confirmed the results from the pH and ionic strength dependencies of the interactions, demonstrating the increased hydrophobic- ity of low-pH buffer-exposed IgG molecules. Fluorescence studies on low-pH buffer-exposed immunoglobulins Here, the physicochemical mechanisms responsible for the increased IgG antigen recognition potential after low-pH buffer exposure were studied. Aromatic amino acids in proteins possess intrinsic fluorescence proper- ties when excited at an appropriate wavelength. The fluorescence characteristics (intensity and wavelength of the fluorescence maxima) depend on the polarity of the local protein environment. Thus, changes in the positions of the fluorescent amino acids, caused by structural modifications of the molecule, result in changes in the microenvironment of the aromatic amino acids that affect the fluorescence characteristics, especially the position of the emission maxima [15,19]. For these reasons, fluorescence spectroscopy is widely used for the analysis of structural changes and of the stability of proteins. We used tryptophan fluorescence in order to deter- mine whether the exposure of immunoglobulin Fig. 4. Low-pH buffer exposure of IgG antibodies results in an enhanced role of the hydrophobic effect in their antigen binding. (A) A pH-scanning ELISA analysis of the interaction of the mouse monoclonal Z2 IgG antibody with its target antigen. (B) The same antigen– antibody interaction in the presence of increasing concentrations of NaCl. In both experiments, binding intensities are represented in RU, and each data point shows the mean value ± standard deviation of quadruplicate wells. Solid lines, native Z2; dotted lines, low-pH buffer- exposed Z2. (C) Emission spectra of 32 l M ANS in the absence or presence of 2 lM native, pH 4 buffer-exposed or pH 2.8 buffer-exposed IVIg. (D) Comparison of the fluorescence characteristics of increasing concentrations of ANS (1–32 l M) in the presence of 2 lM native IVIg (solid lines) or pH 2.8 buffer-exposed IVIg (dashed lines). The emission spectra of ANS were recorded after excitation at 388 nm. I. K. Djoumerska-Alexieva et al. Molecular modifications in low pH-exposed IgG FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3043 molecules to an acidic milieu would result in structural modifications. Indeed, an increase in the fluorescence intensity of the pooled IgG preparation and a slight red shift in the emission maxima following its exposure to a pH 2.8 buffer were detected by fluorescence spec- troscopy (Fig. 5). This effect is consistent with a change in the positions of tryptophan(s) in the immu- noglobulins. The red shift in the emission maxima is indicative of the relocation of tryptophan(s) to a more polar environment. In contrast, treatment of the same preparation with a pH 4 buffer did not change the tryptophan fluorescence of the same molecules. Increase in the relative functional antigen-binding affinity of a monoclonal antibody after its exposure to a pH 4 buffer The relative functional affinities of the native and the low-pH buffer-exposed monoclonal Z2 antibody were analyzed by thiocyanate elution ELISA [20]. In the elution assay, 0–3.0 m potassium thiocyanate was used to disrupt the binding of the Z2 antibody to its target, mouse IgG 2a . The functional affinity was defined by the molar concentration of potassium thiocyanate required for a 50% reduction in binding as detected in ELISA at A 405 nm . The modified mouse monoclonal IgG antibody bound more strongly to the immobilized antigen (Fig. 6). Passive immunotherapy with low-pH buffer-exposed, but not with native IVIg has protective activity in mouse sepsis The effect of treatment with low-pH buffer-exposed IVIg on the survival of lipopolysaccharide (LPS)- injected animals was studied. The decision to test the modified immunoglobulin preparation in experimental sepsis was based on its enhanced binding to IFN-c,on the known role of polyreactive antibodies in infections [10], and on the hypothesis of Antonio Coutinho and Stratis Avrameas suggesting that polyreactive antibod- ies may represent a buffering system that prevents brisk changes in the levels of components of inflamma- tion, coagulation, and other pathways [21]. The admini- stration of a single dose of 500 mgÆkg )1 of the modified IVIg had significant (P < 0.05) protective activity in this experimental sepsis model. The native IVIg was not protective, regardless of the dose used (Fig. 7A–D). Two sets of data strongly argue that the therapeutic effect of the modified IVIg was not due to better neutralization of the injected LPS: first, the binding of the preparation to LPS in its two forms was identical (tested by ELISA, data not shown); and second, the pH 4.0 buffer-exposed IVIg significantly (P < 0.05) decreased mortality, even if injected after the administration of LPS (Fig. 7E). Discussion The brief exposure of polyclonal and at least some monoclonal IgG preparations to a low-pH buffer results in an alteration of the immunoglobulin struc- ture, and in the acquisition of enhanced antigen recog- nition behavior and new biological activities. Our data show that the changes fall short of denaturation of the immunoglobulin molecules. The main argument in support of this claim that low-pH-modified IgG molecules fully retain their F(ab¢) 2 -dependent and Fig. 5. Increase in the fluorescence intensity of pooled IgG after low-pH buffer exposure. Spectrofluorometric analyses of native (solid line), pH 4 buffer-exposed (dotted line) and pH 2.8 buffer- exposed (dashed line) IVIg. The ordinate represents fluorescence intensity in RU. Fig. 6. Increased functional affinity of a mouse monoclonal Z2 anti- body after its exposure to a pH 4 buffer. Thiocyanate elution ELISA was performed as described in Experimental procedures. The func- tional affinity is represented by the molar concentration of potas- sium thiocyanate required for a 50% reduction in binding as detected at A 405 nm . The results represent the average of at least three independent measurements, with the standard deviation indi- cated by error bars (gray bar, native Z2 antibody; black bar, pH 4 buffer-exposed Z2 antibody). Molecular modifications in low pH-exposed IgG I. K. Djoumerska-Alexieva et al. 3044 FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS Fc-dependent antibody functions is based on clinical experience over many years with IVIg preparations produced using a low-pH buffer fractionation step. This study supports a previous suggestion [8] that com- mercially available immunoglobulin preparations are not equal, and shows that the differences between them might be large enough to be clinically relevant. The possibility that nonspecific aggregation of IgG on surface-adsorbed antigens is responsible for the observed enhanced immunoreactivity can be ruled out by the results presented above. Spectroscopic data (tryptophan fluorescence and ANS fluorescence) did not imply the presence of aggregation of the IgG mole- cules in solution after low-pH buffer treatment. In contrast, we observed an increase in the fluorescence signal after exposure of IVIg to pH 2.8 buffer. In the case of IgG aggregation, a decrease in fluorescence intensity would be expected. Nonspecific aggregation on the surface of the adsorbed antigens is also ruled out by the fact that the increased antigen recognition is not observed in the case of all studied antigens. Our real-time kinetic measurements imply that the complex of low-pH buffer-treated IgG with CRP dissociates, although at a slow rate. Wymann et al. [22] have recently suggested that the increased antigen-binding polyreactivity of pH 4 buf- fer-exposed immunoglobulin preparations was mainly caused by the dissociation of IgG dimers in them. Our data strongly suggest, however, that the effect of this exposure goes beyond IgG–IgG dimer dissociation, and affects the IgG molecules themselves. A possible explanation for the finding of new anti- gen-binding specificities after exposure to low-pH con- ditions is the induction of structural rearrangements in the variable region of the antibody. Indeed, we observed changes in the tryptophan fluorescence char- acteristics of IVIg after its exposure to a low-pH (2.8) buffer. The increase in the fluorescence intensity and the red shift in the emission maximum are signs of a change in the position of tryptophan(s) in the IgG molecules – a mark of the existence of a structural modification in the polypeptide chains of the immuno- globulins. In addition, our results revealed that the interactions of low-pH buffer-exposed IgG are less dependent on changes in the ionic strength or the pH of the medium. Such binding behavior is typical of protein–protein bonds that rely on nonpolar types of interaction (hydrophobic effect and van der Waals contacts). Indeed, the increase in the hydrophobicity may well be explained by exposure of previously bur- ied hydrophobic amino acids to the solvent, as shown previously for IgG treated with chaotropic agents [23]. By using the fluorescence molecular probe for hydro- phobicity of proteins (ANS), we confirmed that the exposure of IgG to a low-pH buffer results in molecu- lar modifications characterized by a considerable increase in their surface-exposed hydrophobicity. The antigen-binding behavior of low-pH buffer-exposed IgG preparations was enhanced for some, but not all, antigens tested (e.g. IgG Fc fragments). We are cur- rently investigating whether the proteins that are pref- erentially recognized by the modified antibodies share any common features. It has been observed that the increased polyreacti- vity of a monoclonal IgG after transient exposure to urea correlates well with the elevated flexibility of the antigen-binding site and the involvement of hydropho- bic interactions as a driving force for the recognition of the target antigen. All of these findings allow us to hypothesize that elevated binding to various molecular patterns of monoclonal and polyclonal IgG after their transient exposure to low pH (pH 4 or less) may well be caused by an augmentation of the structural A CD E B Fig. 7. Treatment with pH 4 buffer-exposed IVIg reduces mortality in bacterial LPS-induced septic shock. Survival curves of mice (15 per group) injected with 0.5 mg of LPS and treated with 4 mgÆkg )1 (A), 20 mgÆkg )1 (B), 100 mgÆkg )1 (C) or 500 mgÆkg )1 (D) native IVIg (solid lines), or pH 4 buffer-exposed IVIg (dashed lines), or NaCl ⁄ P i (pH 7.4) alone (gray lines). The protective activity of the modified IVIg is retained, even when its administration has been postponed for 1 h (E). *P < 0.05, Mann–Whitney test. I. K. Djoumerska-Alexieva et al. Molecular modifications in low pH-exposed IgG FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3045 plasticity of their paratopes. Urea and low-pH buffer exposure are both known to induce some degree of melting of the protein conformation. The increased poly- reactivity observed could well be explained by limited melting of immunoglobulin molecules by either of these agents. Our previous data have indicated that treatment of some IgG antibodies with different redox-active agents is also able to enhance antigen-binding polyreactivity by modulating the properties of the antigen-combining sites of some, but not all, studied IgG antibodies. The binding behavior of some, generated in response to repeated immunizations and expected to have rigid, high-affinity binding sites, is not modified by exposure to these conditions [5]. The precise mechanisms that make an individual IgG molecule resistant to polyreac- tivity-inducing treatment remain to be determined. Redox-active agents (heme, iron ions, reactive oxygen species) are released in vivo in inflammation sites as well as after trauma, hemorrhages, etc. Exposure to low pH is part of the production process for some commercial IVIg preparations, and is used on a daily basis for IgG immunoaffinity purification. The dramatically increased antigen-binding polyreac- tivity of polyclonal and of some monoclonal IgG anti- bodies that have been briefly exposed to a pH 4.0 or pH 2.8 buffer suggests that conclusions from many previous studies on IgG, purified by low-pH buffer elution from Protein A, Protein G or other immuno- affinity columns, have to be carefully re-examined. We [24,25] and others [26] have previously used a similar approach to immunopurify anti-self-antigen-binding IgGs from IVIg by recirculating the pooled immuno- globulin preparation through immunoaffinity columns, containing pure immobilized self-antigens, and then eluting the bound fractions by washing the columns with a pH 2.8 buffer. The latter were found to be enriched in natural autoantibodies with the expected specificities. These antibodies were further tested in various in vitro assays, and shown to engage in biologi- cally relevant interactions. Spalter et al. claimed that all individuals, regardless of their ABO histo-blood antigen groups, possessed both anti-A and anti-B anti- gen IgG in their sera. Their main argument was based on the ability of pure IgG, isolated from the sera of donors with an A, B, AB or a O blood group, to bind to the A as well as to the B antigens. The pure IgG fraction was obtained, however, by pH 3 buffer elution from a Protein G Sepharose affinity column. We pro- pose an alternative explanation for the same observa- tions. Even a brief exposure to a low-pH milieu (pH 4 or lower) modifies some of the circulating IgG molecules, resulting in their enhanced antigen-binding polyreactivity and possibly in the acquisition of the ability to bind to a polysaccharide self-antigen (A or B). The low-pH buffer elution of the same fraction may also enhance its capacity to engage in F(ab¢) 2 ⁄ F(ab¢) 2 (idiotype ⁄ anti-idiotype) interactions with other immunoglobulin molecules (see Fig. 2). IVIg preparations are used in patients with primary and secondary immunodeficiencies, as well as in an increasing number of autoimmune and inflammatory diseases. To the best of our knowledge, no compara- tive clinical studies on the immunomodulatory effects of IVIg preparations produced by different protein fractionation technologies have been performed so far. Data from this and from an earlier study [8] strongly suggest that licensed therapeutic IVIgs exposed to pro- duction steps at low pH do acquire new, clinically rele- vant, properties. Their use could be beneficial in the early stages of sepsis, which are characterized by uncontrolled production of proinflammatory mediators (‘cytokine storm’). Previous studies have shown that binding to bacterial LPS is not affected in IgG mole- cules modified by protein-destabilizing agents [5], strongly suggesting that the prevention of LPS-induced sepsis death is due to the ability of this preparation to attenuate the hyperreactivity of body defense mechanisms. In addition to sepsis, there is an increasing number of emerging infectious diseases in which the severe gen- eralized inflammatory reaction of the infected host is a major factor in the poor outcome. Recent additions to the list are H5N1 influenza (avian flu), dengue, Marburg and Lassa hemorrhagic fevers, and West Nile virus infection [27–31]. One could speculate that pas- sive immunotherapy with ‘modified’ IVIg preparations would be beneficial in patients with these diseases. An important argument in favor of low-pH buffer-exposed IVIg is that it has already been in clinical use for a long time, whereas the ferrous ion and heme-exposed IVIg preparations are at an early preclinical evaluation stage. Experimental procedures Monoclonal antibody immunoglobulin preparations, and immunoglobulin fragments The Z2 hybridoma producing a mouse monoclonal IgG 2b antibody against mouse IgG 2a was kindly provided by E. Rajnavolgyi (Department of Immunology, Lorand Eotvos University, Budapest, Hungary). The commercial intrave- nous immunoglobulins Endobulin S ⁄ D (Baxter, Deerfield, IL, USA), and Octagam (Octapharma, Lachen, Switzerland) were used in the experiments. F(ab¢) 2 and Fc fragments of Molecular modifications in low pH-exposed IgG I. K. Djoumerska-Alexieva et al. 3046 FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS IVIg, as well as an experimental pooled human IgM preparation, were prepared as described previously [32,33]. Pure human Fc-l fragments, obtained from a patient with l-heavy chain disease, were a gift from L. Mouthon (Cochin Hospital, Paris). The Z2 antibody and IVIg samples were diluted in 0.1 m sodium acetate buffer (pH 4.0 or 2.8) and incubated for 5 min. The pH was then brought to 7.0, and the samples were dialyzed against NaCl ⁄ P i (pH 7.2) [8]. The pH 4.0 buffer was chosen because several commercial IVIg prepa- rations are produced using a fractionation step with this pH value. Buffers of pH 2.8 are widely used for the isola- tion of pure IgG by affinity chromatography. Immunoblot analysis The total lysate from a nonpathogenic strain of B. anthra- cis was kindly provided by S. Mesnage (Centre de Recher- che des Cordeliers, Paris, France). A lysate of E. coli was prepared as described elsewhere [5]. Both bacterial antigen extracts were subjected to 10% SDS ⁄ PAGE and trans- ferred to nitrocellulose membranes (Scheicher & Schuell, Dassel, Germany) with a Mini Transfer Blot system (Bio- Rad, Richmond, CA, USA) in a buffer containing 48 mm Tris, 110 mm glycine, and 20% (v ⁄ v) methanol. Then, they were incubated for 1 h at room temperature in NaCl ⁄ Tris containing 0.3% Tween-20. Membranes were further cut into strips or fixed in a miniblot system, and incubated for 1 h at room temperature with the native, the pH 4.0 buffer-exposed or the pH 2.8 buffer-exposed IVIg prepara- tions (at 0.1 mgÆmL )1 ). After extensive washing, they were incubated with goat anti-human IgG (Fc-specific), conju- gated to alkaline phosphatase (Southern Biotech, Birming- ham, AL, USA), and finally developed using the Nitro Blue tetrazolium and bromo-chloro-indolyl-phosphate substrates (both from Sigma-Aldrich, Taufkirchen, Germany). The quantitation of bound antibodies in immu- noblots was performed by densitometry in reflective mode, using a UMAX 1220p scanner linked to a PC. The data were analyzed using imagetool v2.0 for Windows (UTHSCSA, San Antonio, TX, USA). Migration distance (x-axis) was expressed in pixels, and intensity of binding level (y-axis) was expressed in relative units (RU). ELISA Ninety-six-well polystyrene plates (Brand GMBH, Wert- heim, Germany, or Nunc, Denmark) were coated with 5 lgÆmL )1 recombinant human IFN-c (a gift from I. Iva- nov, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria), with 2 lgÆmL )1 human fac- tor VIII, 10 lgÆmL )1 human factor IX (LFB, France), 10 lgÆmL )1 human CRP, 10 lgÆmL )1 human C3 (both from Calbiochem), 10 lgÆmL )1 human factor H, 10 lgÆmL )1 human factor B (both from Complement Technology, TX, USA), 20 lgÆmL )1 porcine thyroglobulin, 20 lgÆmL )1 rabbit tubulin, and 10 lgÆmL )1 bovine myelin basic protein (all three from Sigma-Aldrich), for 2 h at room temperature. Plates were blocked with 0.25– 0.4% (v ⁄ v) Tween-20 in NaCl ⁄ P i for 2 h. After washing with NaCl ⁄ P i containing 0.05% Tween-20, the plates were incubated overnight at 4 °C (in the case of IFN-c) or for 2 h at 25 °C (in the case of other proteins) with increasing concentrations of the immunoglobulin preparations under study. The plates were then extensively washed, and goat anti-(human IgG) (c-chain-specific) coupled to alkaline phosphatase was added and incubated for 1 h at room tem- perature. Immunoreactivities were revealed by adding p-ni- trophenyl phosphate (Sigma-Aldrich) diluted in appropriate buffers. The pH and salt concentration dependence of the binding of the native or low-pH buffer-exposed mouse monoclonal Z2 antibody to its cognate antigen were analyzed by ELISA. Polystyrene plates were coated with 20 lgÆmL )1 of a mouse IgG 2a monoclonal antibody (clone IP2-11-1). The free binding sites were blocked with NaCl ⁄ P i containing 0.5% (v ⁄ v) Tween-20 for 2 h at room temperature. After washing, the plates were incubated overnight at 4 °C with dilutions of the native or the low-pH buffer-exposed Z2. For the pH-scanning analysis, the Z2 antibody was diluted to 15 lgÆmL )1 in buffers with different pH values, as fol- lows: in citrate ⁄ phosphate-buffered saline [0.05 m sodium citrate, 0.05 m Na 2 HPO 4 , 0.14 m NaCl, 0.05% (v ⁄ v) Tween-20] with a pH in the range 3–7.5, or in carbonate- buffered saline [0.05 m NaHCO 3 , 0.05 m Na 2 CO 3 , 0.14 m NaCl, 0.05% (v ⁄ v) Tween-20] with a pH in the range 8.5–12. In the ELISA with increasing salt concentrations, the Z2 antibody was diluted to 15 lgÆmL )1 in buffers with 0–4 m sodium chloride. After a 2 h incubation step under the described conditions, the plates were washed and further incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG 2b (PharMingen, San Diego, CA, USA) for 1 h at room temperature. The following steps of the assay were performed as described above. The results were represented in RU. The binding at pH 7.0 or in the presence of 0 m NaCl buffers, respectively, was referred to as 1 RU. The abilities of both IVIg variants to engage in idio- type ⁄ anti-idiotype interactions were compared by ELISA. Polystyrene plates were coated with F(ab¢) 2 or Fc IVIg fragments, with pooled IgM, or with pure Fc-l fragments (all at 10 lgÆmL )1 in coating buffer). The blocking and washing steps were performed as described above. The plates were incubated with increasing concentrations of the IVIg preparations under study, and after extensive washing, goat anti-human IgG (Fc-specific; Sigma-Aldrich) coupled to alkaline phosphatase was added for an additional 1 h at room temperature. In the case when Fc-c fragments were used as coating antigen, goat anti-human IgG [F(ab) 2 -spe- cific] (PharMingen, San Diego, CA, USA) was used to I. K. Djoumerska-Alexieva et al. Molecular modifications in low pH-exposed IgG FEBS Journal 277 (2010) 3039–3050 ª 2010 The Authors Journal compilation ª 2010 FEBS 3047 reveal antibody binding. The final steps were performed as described above. Real-time kinetic measurements The kinetic constants of the interactions between IVIg and human CRP were determined by surface plasmon resonance (BIAcore 2000; GE Biacore, Uppsala, Sweden). CRP was immobilized on research-grade CM5 chips, using an amino- coupling kit (Biacore) as described by the manufacturer. In brief, CRP was diluted in 5 mm maleic acid (pH 5) to a final concentration of 100 lgÆmL )1 , and coated on the pre- activated sensor surface. Experiments were performed using HBS-EP (0.01 m Hepes, pH 7.4, containing 0.15 m NaCl, 3mm EDTA, and 0.005% Tween-20) as running and sam- ple dilution buffer. IVIg (native or low-pH buffer-exposed Endobulin) was injected at concentrations in the range 5–0.039 lm at a flow rate of 10 lLÆmin )1 . The association and dissociation phases of the interaction were monitored for 5 min. The regeneration of the chip surface was performed using a 5 m solution of guanidine-HCl (Sigma- Aldrich). The binding to the surface of the uncoupled control flow cell was always subtracted from the binding to the protein-coated flow cells. biaevaluation software (ver- sion 4.1; Biacore) was used for the calculation of the kinetic rate constants. Calculations were performed by global analysis of the experimental data using the kinetic models included in the software, fitting the data with lowest value of v 2 . Fluorescence spectroscopy Intrinsic emission spectra measurements of the native and of the low-pH buffer-exposed IVIg were performed on a Hitachi F-2500 spectrofluorometer. Samples of the IVIg were exposed at 4 °C to pH 4 or pH 2.8 acetate buffers. After incubation, the samples were dialyzed against NaCl ⁄ P i . All analyses were carried out at 25 °C, using 1 cm quartz cuvette. The samples were diluted in NaCl ⁄ P i to a final concentration of 2 lm. A wavelength of 295 nm, which excites tryptophans, was used for the fluorescence spectra measurements. The fluorescence emission spectra were recorded between 300 and 450 nm. The excitation and emission slits were both 10 nm, and the scan speed was 1500 nmÆmin )1 . ANS fluorescence ANS was obtained from Sigma-Aldrich. IVIg prepara- tions (at 2 lm, native as well as low-pH buffer-exposed) were mixed with increasing concentrations of ANS (1–32 lm). After excitation at 388 nm, the fluorescence emission spectra of ANS were recorded between 425 and 600 nm in a 1 cm quartz curette. The excitation and emission slits were set to 10 nm, and the scan speed was 1500 nmÆmin )1 . Thiocyanate elution ELISA The thiocyanate elution ELISA was performed as described previously [20]. Briefly, ELISA plates were coated with a mouse IgG 2a monoclonal antibody and, after washing, were further incubated overnight at 4 °C with 15 lgÆmL )1 of the native or low-pH buffer-exposed mouse monoclonal Z2 antibody in the presence of increasing concentrations of potassium thiocyanate (ranging from 0 to 2.0 m). After incubation and washing, the antibody binding was mea- sured using a goat anti-mouse IgG 2b as described above. Experimental septic shock Outbred ICR mice were purchased from the Breeding Farm of the Bulgarian Academy of Sciences. The experimental protocols were approved by the Animal Care Commission of the Institute of Microbiology, in accordance with National and European Regulations. The number of ani- mals used was kept at the minimum that still ensured statis- tical significance of survival differences between the experimental groups. Septic shock was induced in 16–18- week-old animals by the intraperitoneal administration of 400 lg of bacterial LPS (from E. coli B 055:B5, Sigma- Aldrich, #L2880). Minutes later, groups of mice (15 per group) were injected intravenously with increasing doses of the native IVIg or of the low-pH buffer-exposed IVIg prep- aration, or with NaCl ⁄ P i alone. Survival was observed for 5 days. In a separate experiment, the native and the modi- fied IVIg (500 mgÆkg )1 , 10 animals per group) were infused 1 h after the administration of LPS. Any effect of a treat- ment started at this time-point should be the result of its influence on the pathophysiological mechanisms of the sep- sis syndrome [34]. Statistical analysis Statistical analyses were performed using graphpad prism, version 4.00 (GraphPad Software, San Diego, CA, USA). Statistical analyses of the ELISA data and the areas under the curve of the immunoblot densitometric profiles were performed using the paired Student t-test. For survival analyses, differences between groups were analyzed by the Mann-Whitney U-test. In all cases, P-values < 0.05 were considered to indicate statistical significance. Acknowledgements This study was supported by the Bulgarian National Science Fund (grants VU-L-314 ⁄ 07 and TK-X- 1710 ⁄ 07), by the NATO Science for Peace Program Molecular modifications in low pH-exposed IgG I. K. 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