Histidine-rich glycoprotein regulates the binding of monomeric IgG and immune complexes to monocytes

Nick N. Gorgani, Joseph G. Altin1 and Christopher R. Parish

Division of Immunology and Cell Biology, The John Curtin School of Medical Research, and
1 Division of Biochemistry and Molecular Biology, School of Life Sciences, Faculty of Science, Australian National University, Canberra, ACT 0200, Australia

Correspondence to: C. R. Parish


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein which we have shown previously inhibits the formation of insoluble immune complexes (IC). In this study we examined the ability of HRG to regulate the binding of monomeric IgG and IC to monocytes. Initial studies demonstrated that HRG interacts with Fc{gamma}RI on the monocytic cell line THP1 and blocks the binding of monomeric IgG to these cells. However, despite totally blocking the binding of monomeric IgG to Fc{gamma}RI, pre-incubation of THP1 cells with HRG had no effect on the binding of IC to these cells. In contrast, depending on the HRG:IgG molar ratio, pre-incubation of monomeric IgG with HRG resulted in either enhanced or reduced IgG binding to Fc{gamma}RI. Similarly, under certain highly defined conditions, incorporation of HRG in IgG-containing IC potentiated the binding of IC to THP1 cells. The key conditions involved incorporating approximately equimolar concentrations of HRG and IgG in the IC, the IC being formed at a near equivalence antigen:antibody ratio and usually physiological concentration (20 µM) of Zn2+ being present. Collectively these observations indicate that HRG is an important regulator of IC uptake by monocytes. Thus HRG can interact with Fc{gamma}RI on monocytes and block monomeric IgG binding, whereas when incorporated in IgG containing IC, HRG can enhance the uptake of IC by monocytes, probably via its heparan sulfate binding domain.

Keywords: Fc{gamma}RI, histidine-rich glycoprotein, IgG, immune complex, monocytes/macrophages


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The recognition of a cognate antigen by polyclonal antibody molecules in the blood circulation may result in the formation of soluble and insoluble immune complexes (IC). The mechanisms by which such IC are cleared from the circulation and from tissues are not well understood, but the binding of IC to FcR (1,2) and, following complement activation, to complement receptors (CR) (3,4) on mononuclear phagocytic cells is known to be important. Three different types of FcR specific for IgG, i.e. Fc{gamma}RI, Fc{gamma}RII and Fc{gamma}RIII (each similar in biological function but capable of binding distinct IgG subclasses), are known to exist in humans (5,6). Evidence suggests that the binding of IgG to FcR on mononuclear cells can trigger a number of responses including the phagocytosis, uptake and clearance of IC. Thus, under normal physiological conditions IC may be recognized by cells of the reticuloendothelial system (RES) such as peripheral blood mononuclear phagocytes (1,7,8) and resident phagocytic cells in liver and/or spleen and cleared from the circulation (3,9,10). Similarly, in many tissues IC may be cleared by tissue macrophages and monocytes which are recruited from the circulation as part of the inflammatory response (4).

A role for components of complement in the uptake and clearance of IC by phagocytic cells was first suggested by the demonstration that receptors for C3 exist on monocytes (11), and that large IC in blood can activate complement and both generate and incorporate C3 fragments such as C3b and C3d (12). Subsequent studies showed that blood borne C3b-opsonized IC are usually recognized by C3b receptors (CR1) on erythrocytes (3,10,13). Erythrocytes, upon entering the liver and/or spleen (14,15) may release bound IC (16) which are then free to interact with resident tissue mononuclear cells such as macrophages and monocytes via receptors for Fc, C3b and C3d (9,17,18).

Histidine-rich glycoprotein (HRG) is a plasma protein of ~80 kDa that has been shown to bind heparin (19), plasminogen (20), fibrinogen (21) and divalent metal ions (22,23), and to regulate aspects of the immune system (2428). In recent studies we also showed that HRG binds to IgG-containing soluble IC and inhibits the formation of insoluble IC in vitro by promoting the maintenance of IC in a soluble form (29). HRG also is reported to bind to macrophages, and to regulate FcR expression and phagocytosis (27,30). Since HRG incorporates in IC and binds to macrophages, it was important to examine whether HRG plays any role in the clearance of IC by mononuclear phagocytes. The present work uses flow cytometry to study the effect of HRG on the binding of IgG and IC to the human mononuclear phagocytic cell line THP1. The results show that HRG inhibits the binding of monomeric human or rabbit IgG to Fc{gamma}RI on THP1 cells, but promotes the binding of IC containing either human or rabbit IgG to these cells in a Zn2+-dependent manner. Collectively, the results indicate that HRG regulates the binding of both monomeric IgG and IC to mononuclear cells, and provide evidence for the existence of a complement-independent mechanism for enhancing the clearance of IC from the circulation.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
Human IgG (from pooled human plasma), human IgG subclasses (myeloma derived), BSA (fraction V), ovalbumin (OVA; grade V) and DMSO were purchased from Sigma (St Louis, MO). FITC-conjugated streptavidin (STP) was purchased from Amersham (Little Chalfont, UK. The mAb against Fc{gamma}RI (mAb22) was provided by the organizers of the Fifth Leukocyte Differentiation Antigen Workshop.

Purification of proteins
Native human HRG of 80 kDa mol. wt. was purified from fresh human plasma as previously described (24) by equilibrating a phosphocellulose column with loading buffer comprising 10 mM sodium phosphate (pH 6.8) containing 1 mM EDTA and 0.5 M NaCl (loading buffer). The plasma was mixed with EDTA and NaCl at the final concentrations as in the loading buffer and with [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] (ICN, Costa Mesa, CA) at 100 µg/ml and aprotinin (a trypsin inhibitor) at 2 µg/ml. The plasma was passed through the equilibrated column and unbound protein was removed by extensive washing of the column with the loading buffer. Bound HRG was then eluted from the column using the same buffer containing 2 M NaCl.

Rabbit anti-OVA IgG was purified from immunized rabbit serum by Na2SO4 precipitation and ion-exchange chromatography using a diethylaminoethyl Sephacel column. The IgG was collected from the break through peak as described (31). Using SDS–PAGE analysis the purity of the HRG and IgG preparations as judged by the intensity of the contaminating bands was always >95% (29). In some experiments the purified IgG was depleted of any IgG aggregates by passage through a FPLC Superose-12 column (Pharmacia, Uppsala, Sweden), and the monomeric IgG fraction (~150 kDa) collected and used immediately.

Formation of insoluble IC
The formation of insoluble OVA:anti-OVA IgG IC was carried out by incubating rabbit polyclonal anti-OVA IgG in PBS containing 3 mM NaN3 and different amounts of HRG for 5 min in a quartz reaction vessel (final volume 1 ml) maintained at 37°C. The formation of insoluble IC was initiated by the addition of OVA at different antigen:antibody ratios. Since the absorbance due to light scattering of a suspension of particles is related to the average size of the particles, the formation of insoluble IC was monitored by measuring the absorbance of samples at 350 nm using a Varian Cary-1 spectrophotometer as described (29).

Precipitin curve analysis of IC containing rabbit IgG and OVA
Anti-OVA IgG (1 mg/ml) was mixed with different concentrations of OVA at antigen:antibody ratios in the range of 0.005–0.06 (w/w) in PBS containing 10 mg/ml BSA (PBS/BSA) and 3 mM NaN3 (PBS/BSA/Az) to a final volume of 1 ml and incubated overnight at 37°C. The insoluble IC in each suspension were collected by centrifugation (104 g, 1 min), washed 3 times in PBS and then dissolved in 0.2 M NaOH. The extent of IC formation was then calculated by measuring the absorbances of each solution at 280 nm, assuming E 0.1% (w/v) = 1.4 for IgG.

FITC conjugation of IgG
Purified rabbit anti-OVA IgG (2 mg/ml) was equilibrated with coupling buffer by extensive dialysis against 0.05 M boric acid, 0.2 M NaCl (pH 9.2), and then 50 µl/ml of FITC (5 mg/ml) (Molecular Probes, Eugene, OR) dissolved in DMSO was added and the mixture incubated for 2 h at room temperature in the dark. Unreacted FITC was removed by subjecting the sample to five cycles of concentration and dilution in an Amicon ultrafiltration apparatus (Amicon, Beverly, MA). The FITC–IgG was sterile filtered using a DynaGard 0.2 µm filter (Microgon, Laguna Hills, CA), before storing at 4°C until use. Based on fluorometer measurements it was estimated that approximately four molecules of FITC were coupled to each IgG molecule.

Biotinylation of proteins
HRG and human IgG were dissolved in PBS (pH 7.2) at a concentration of 1 mg/ml and then reacted with NHS-LC-biotin (1 mg/ml) (Pierce, Rockford, IL) for 30 min at room temperature. The reaction was stopped by the addition of Tris–HCl buffer (pH 8.0) to a final concentration of 100 mM. Unreacted biotin was removed by washing the sample extensively (five cycles of concentration and dilution) in a Centricon 10 microconcentrator, before storing the biotinylated proteins at –20°C in small aliquots until use. Based on a competitive ELISA assay it was estimated that HRG and IgG were conjugated with approximately six and four molecules of biotin/molecule respectively.

Cell culture
The human THP1 and U937 monocyte cell lines, human Jurkat cells (CD4+ leukemic T cell line), and human MT4 cells (CD4+ T cell line) were all cultured in RPMI 1640 (Gibco, Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (Commonwealth Serum Laboratories, Melbourne, Australia) and antibiotics (penicillin 120 µg/l, streptomycin 200 µg/l and neomycin 200 µg/l) at 37°C in a 5% CO2 atmosphere. The cell lines were routinely subcultured at ~0.25x106 cells/ml and allowed to grow to the maximum concentration of ~1x106 cells/ml.

Immunofluorescent flow cytometry
The cultured cells were transferred to plastic 50 ml Falcon tubes (Becton Dickinson, Lincoln Park, NJ) and centrifuged at 450 g for 5 min. Supernatants were discarded and the cells were washed 3 times in PBS. The concentration of cells was determined using a hemocytometer, and the cell suspensions pelleted and resuspended in PBS/BSA or PBS/BSA containing 20 µM Zn2+ (PBS/BSA/Zn) buffer to a final concentration of 1x107 cells/ml. The cell suspensions were then pipetted (25 µl/well) into each well of a 96-well V-bottomed plastic plate (Nunc, Roskilde, Denmark), human HRG was added at different concentrations (25 µl/well) and the mixture incubated for 1 h on ice. The cells were washed 3 times by centrifugation (800 g, 1 min) with the appropriate buffer to remove the unbound HRG, and biotinylated or FITC–IgG conjugate was added in the appropriate buffer and incubated with the cells for 1 h on ice. The cells were washed 3 times by centrifugation and bound biotinylated IgG was detected by incubating the cells with FITC–STP (~ 50 µg/ml). After removing unbound FITC–STP by centrifugation, the cells were resuspended in PBS/BSA or PBS/BSA/Zn buffer (50 µl/well) and fixed by the addition of 4% paraformaldehyde in PBS (50 µl/well).

The fluorescence intensity of the cells was measured by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA). In some experiments cell populations were gated, according to the forward and side scatter, to eliminate the fluorescence deriving from dead cells, aggregated cells or large insoluble IC. The results obtained for each sample were analysed by the CellQuest program. Using this program, the median fluorescence intensity units (FIU) of each fluorescence intensity histogram were determined and plotted against the concentration of ligand or inhibitor used.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Binding of human IgG and HRG to THP1 cells
It is well established that monomeric IgG binds to monocytes and macrophages through Fc{gamma}RI on the surface of these cells (7,8,32,33). Previous studies also have shown that HRG binds to peritoneal macrophages and regulates phagocytosis of antibody opsonized erythrocytes (27,30). Initial experiments were aimed at determining the level of binding of human IgG and HRG to the monocytic cell lines THP1 and U937 by flow cytometry. For these studies the cells were incubated with different concentrations of biotinylated human IgG (bIgG) or HRG (bHRG) in PBS/BSA, washed and then stained with FITC–STP. The data in Fig. 1Go show a plot of the median fluorescence intensity (Medium FIU) of the cells against different bIgG (Fig. 1AGo) or bHRG (Fig. 1BGo) concentrations, and indicate that binding of both IgG and HRG to THP1 cells is concentration dependent. Similar binding curves were obtained when U937 cells were used instead of THP1 cells (not shown).



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Fig. 1. Binding of human IgG or HRG to THP1 cells as measured by fluorescent flow cytometry. In (A) and (B) the binding of different concentrations of biotinylated IgG or HRG to THP1 cells is depicted, the binding of each molecule being detected with FITC–STP and the data being expressed as median FIU values. Each of the data points are representative results obtained from three separate experiments. In (C) the effect of heparin (50 µg/ml), IgG (2 µM) and a Fc{gamma}RI-specific mAb (mAb22; saturating concentration) on the binding of bHRG (2 µM) to THP1 cells is shown. In this experiment HRG was pre-incubated with heparin prior to the addition of the THP1 cells or THP1 cells were pre-incubated with IgG or the mAb22 prior to HRG addition. Each histogram represents the mean ± SEM of three determinations, with P values relative to the no inhibitor control being shown in the figure.

 
Additional experiments indicated that pre-incubation of bHRG (2 µM) with heparin (50 µg/ml) significantly inhibited the binding of bHRG to THP1 cells by ~60%, whereas pre-incubating THP1 cells with 2 µM IgG inhibited bHRG binding by ~35% (Fig. 1CGo), an effect which approached significance. Furthermore, these inhibitory effects were additive: pre-incubation of bHRG (2 µM) with heparin (50 µg/ml) and pre-incubation of THP1 cells with IgG (4 µM) inhibiting the binding of HRG to THP1 cells by 80–90% (Fig. 1CGo), an effect which was highly significant. In addition, pre-incubation of THP1 cells with a saturating amount of a mouse mAb (mAb22) which binds to a region of human Fc{gamma}RI adjacent to the IgG-binding site (34) partially inhibited (35%) the subsequent binding of bHRG to the cells, supporting the notion that HRG interacts with Fc{gamma}RI (Fig. 1CGo). As previously shown for other cell types, the presence of physiological concentrations (20 µM) of Zn2+ potentiated the binding of HRG to THP1 cells by ~45% (data not shown), an effect which has been shown to be due to an enhancement of heparin-inhibitable binding (28, 35). These results are consistent with HRG interacting with at least two different binding sites on THP1 cells: sites inhibitable by heparin (presumably cell surface heparan sulfates) and sites inhibitable by IgG (the cell surface Fc{gamma}RI).

Effect of pre-treating THP1 cells with HRG on monomeric IgG binding
The observation that HRG binds to Fc{gamma}RI on THP1 cells raised the question of whether cell-bound HRG affects the ability of the cells to bind monomeric IgG via Fc{gamma}RI. To explore this possibility, THP1 cells were either pre-incubated with PBS/BSA or pre-incubated with PBS/BSA containing 2, 4 and 8 µM HRG, before washing and incubating the cells with different concentrations (40 pM to 40 nM) of monomeric bIgG. After washing away unbound bIgG the cells were then stained with FITC–STP and subjected to FACS analysis. To ensure that the bIgG was monomeric the bIgG was fractionated on a FPLC Superose-12 gel filtration column, and the monomeric IgG fraction collected and used immediately. Very similar results were obtained with both the unfractionated and the FPLC fractionated IgG preparations.

The data in Fig. 2Go(A) show the median FIU of cells following incubation with different IgG concentrations, and indicate that pre-incubation of THP1 cells with 2, 4 and 8 µM HRG strongly inhibited subsequent binding of IgG. In fact, at the higher concentrations of HRG (4 and 8 µM) an almost total inhibition of IgG binding was observed. Similar experiments also were carried out in which the effect of pre-incubating THP1 cells with different concentrations of HRG (9 nM to 9 µM) on the subsequent binding of a constant concentration of IgG (20 nM) was examined (Fig. 2BGo). The results indicate that the pre-treatment of THP1 cells with HRG inhibits the subsequent binding of IgG and that this inhibition is dependent on the HRG concentration with HRG concentrations >=2 µM inhibiting monomeric IgG binding by 90% or more (Fig. 2BGo, solid squares).



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Fig. 2. Effect of HRG on the binding of monomeric human IgG to THP1 cells as measured by fluorescent flow cytometry as described in Fig. 1Go. (A) The binding of different concentrations of monomeric IgG to control THP1 cells, and to THP1 cells pre-treated with 2, 4 and 8 µM HRG. (B) The effect of pre-incubating either THP1 cells (squares) or monomeric IgG (triangles) with different HRG concentrations on the subsequent binding of monomeric IgG to THP1 cells. Pre-incubations were carried out either in the absence (solid symbols) or presence (open symbols) of 20 µM Zn2+. Each point is a representative result obtained from three separate experiments carried out at each concentration of IgG (A) and HRG (B).

 
Recently we showed that the presence of Zn2+ modulates the binding of HRG to several different T cell lines (28) and to IgG (29). To determine whether Zn2+ also can modulate the effect of HRG on the binding of IgG to THP1 cells, the experiments described above also were performed with 20 µM Zn2+ being included in the binding buffer (PBS/BSA/Zn). The results of these studies indicate that the presence of 20 µM Zn2+ had no significant effect on the binding of IgG to THP1 cells (not shown) and had little or no modulatory action on the inhibitory effect of HRG on IgG binding (Fig. 2BGo, open squares).

Effect of pre-incubating IgG with HRG on monomeric IgG binding to THP1 cells
The ability of HRG to bind IgG (29) suggested that the pre-incubation of IgG with HRG also might influence the binding of IgG to Fc{gamma}RI on THP1 cells. To explore this possibility bIgG was first pre-incubated with different concentrations of HRG in PBS/BSA and the mixture was then incubated with THP1 cells. Binding of the IgG to the cells again was assessed by FACS analysis after staining the cells with FITC–STP. As shown by the plot of IgG binding against HRG concentration (Fig. 2BGo), the pre-incubation of bIgG (20 nM) with relatively low concentrations (~20–280 nM) of HRG enhanced binding of the IgG to THP1 cells, whereas pre-incubation of the IgG with higher concentrations (1.12–9 µM) of HRG partially inhibited binding of the IgG to these cells (Fig. 2BGo filled triangles). Parallel experiments also were carried out in which 20 nM bIgG was pre-incubated with different concentrations of HRG in PBS/BSA/Zn before incubating the mixture with THP1 cells. Overall Zn2+ had little or no effect on the ability of HRG to modulate the binding of IgG to THP1 cells (see Fig. 2BGo, open triangles). Control experiments indicated that Zn2+ alone had no effect on the binding of IgG to THP1 cells (not shown).

The effect of HRG on the binding of IC to THP1 cells
To facilitate studies on the binding of IC to THP1 cells, FITC-labeled monomeric rabbit anti-OVA IgG (FITC–IgG) was prepared and this antibody used to prepare fluorescent IC which could be used for cell binding studies. Preliminary studies showed that monomeric FITC–rabbit IgG bound to THP1 cells in an almost identical manner to monomeric human IgG, i.e. near maximal binding at ~10 nM FITC–IgG (not shown). Also, an analysis of the precipitin curve for IC containing OVA and FITC–anti-OVA IgG indicated that maximum precipitation (~ 38% of the total added FITC–IgG) occurs when the ratio of OVA:FITC–IgG is between 0.02 and 0.04 (Fig. 3AGo, open squares). This ratio is similar to that obtained from an analysis of OVA and non-FITC-labeled anti-OVA IgG containing IC (not shown). These results suggest that the labeling of the anti-OVA IgG with FITC does not significantly affect the ability of the monomeric IgG to bind to THP1 cells or to form IC with OVA.



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Fig. 3. Binding of IC containing FITC-labeled rabbit IgG (FITC–IgG) to THP1 cells as measured by fluorescent flow cytometry. The binding to THP1 cells of fluorescent IC formed at different antigen:antibody ratios is shown in (A). The precipitin curve analysis for the formation of insoluble IC containing FITC–IgG and OVA also is shown in (A), with the percent precipitation of FITC–IgG at each antigen:antibody ratio being depicted. The histograms in (B) represent the FITC fluorescence profiles due to the binding to THP1 cells of OVA–FITC–IgG IC formed either at antigen excess (0.06 antigen:antibody ratio) or at equivalence (0.03 antigen:antibody ratio). The free-IgG line represents the fluorescence profile of cells incubated with 0.5 µM of free FITC–IgG (no OVA added control). The data are representative results obtained from three separate experiments.

 
To study the binding of IC to THP1 cells, different concentrations of OVA were pre-incubated with FITC–IgG (0.5 µM) for 30 min in PBS/BSA buffer to allow the formation of IC and then each mixture was incubated separately with THP1 cells. OVA:FITC–IgG ratios were used to yield IC in antibody excess, antigen excess and at equivalence. After incubation with IC the cells for each IC preparation were washed to remove unbound fluorescent IC and subjected to FACS analysis. The data in Fig. 3Go(B) show that relative to cells incubated with 0.5 µM of free FITC–IgG (control), THP1 cells incubated with IC formed in antigen excess (antigen:antibody ratio = 0.06) showed a homogeneous increase in FITC fluorescence (Fig. 3BGo) as a consequence of the binding of FITC-containing IC to the cells. When THP1 cells were exposed to IC formed at an equivalence antigen:antibody ratio, the binding of fluorescent IC became more heterogeneous and broadly distributed, with some cells showing a very high level of fluorescence while others showed a level of IC binding similar to that seen with IC formed in antigen excess (Fig. 3BGo). This broad distribution in fluorescent IC binding to the cells most likely reflects great heterogeneity in the size of IC formed at equivalence. Figure 3Go(A, circles) clearly shows that IC formed at equivalence or in slight antibody excess bind best to THP1 cells, whereas IC binding is less evident when the complexes are formed in slight antigen excess (Fig. 3A and BGo).

Subsequent experiments investigated the effect of HRG on the binding of fluorescent OVA anti-OVA IC to THP1 cells. Pre-incubating THP1 cells with 2 µM HRG had no significant effect on the binding of IC formed with 0.5 µM FITC–anti-OVA IgG at either equivalence or in antigen excess (not shown). HRG also has no effect when 20 µM Zn2+ was included in the assay. However, as with monomeric human IgG (Fig. 2Go), pre-treatment of the THP1 cells with HRG inhibited the binding of monomeric FITC–anti-OVA IgG, an effect which was observed even when IC were present (not shown).

Our previous results (Fig. 2BGo, triangles) indicate that the binding of human IgG to THP1 cells is enhanced when monomeric human IgG is pre-incubated with HRG (HRG:IgG molar ratio ~1–10:1) before incubating the HRG–IgG mixture with the cells. To determine whether HRG also enhances the binding of IC to THP1 cells, HRG was incorporated in IC by pre-incubating anti-OVA FITC–IgG with HRG prior to the addition of OVA and then incubating the resultant IC with THP1 cells. In the absence of Zn2+ HRG has little or no effect on the binding of IC to THP1 cells at a wide range of HRG concentrations and at different antigen:antibody ratios (not shown). However, inclusion of 20 µM Zn2+ in the assay did result in HRG substantially enhancing the binding of IC to THP1 cells (Fig. 4Go). This potentiating effect of HRG, in the presence of Zn2+, on the binding of IC to THP1 cells was seen when the HRG:IgG molar ratio approached 0.5–1 (Fig. 4AGo). However, at higher HRG:IgG molar ratios little potentiation occurred and, in fact, HRG at a 4-fold molar excess to IgG began to inhibit IC binding (Fig. 4AGo). A similar effect of the HRG:IgG molar ratio on IC binding to THP1 cells was observed when IC were formed over a wide range of antigen:antibody ratios (Fig. 4BGo). Enhancement of IC binding was most marked when 0.25 and 0.5 µM HRG was incorporated in the IC (Fig. 4BGo), with lower HRG concentration (<=0.125 µM) being much less effective at enhancing IC binding (not shown).



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Fig. 4. Effect of the HRG:IgG molar ratio on the binding of IC to THP1 cells. IC were formed between OVA and anti-OVA FITC–IgG (0.5 µM) at different antigen:antibody ratios and in the presence of different concentrations of HRG. Binding of fluorescent IC to THP1 cells was then determined by flow cytometry and the data in (A) presented as relative binding (RB) for each HRG:FITC–IgG molar ratio where RB = [(fluorescence of THP1 cells incubated with HRG treated IC – background THP1 cell fluorescence)/(fluorescence of THP1 cells incubated with HRG untreated IC – background THP1 cell fluorescence)]x100. (A) Effect of the HRG:IgG molar ratio on the binding of IC to THP1 cells formed at antigen:antibody ratios of 0.01, 0.02 and 0.03, the antigen:antibody ratio of 0.03 representing equivalence. (B) Overlay plots of the binding to THP1 cells of IC containing 0.5 µM anti-OVA FITC–IgG formed at different antigen:antibody ratios when IC were either untreated or were pre-treated with 0.25, 0.5, 1 and 2 µM HRG in PBS/BSA/Zn. Experiments in (B) were performed at six different concentrations of HRG in the range of 62.5 nM to 2 µM; however, for clarity, only the data for four concentrations of HRG are plotted against the different antigen:antibody ratios. The data are representative results obtained from three separate experiments.

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study shows that the presence of human HRG markedly affects the binding of free human IgG and IgG-containing IC to the human monocytic cell lines THP1 and U937. Consistent with the known ability of monomeric IgG to bind to Fc{gamma}RI on mononuclear cells (7,8,32,33,36) and the reported interaction of HRG with mouse peritoneal macrophages (27,30), our flow cytometric analyses indicate that human bIgG and human bHRG each binds to THP1 cells in a concentration-dependent manner with near-maximal binding of the IgG to THP1 cells occurring at a concentration of 20 nM IgG (Fig. 1AGo), a result similar to that reported in previous studies (7,37,38). Of particular interest, however, was the finding that HRG binds to Fc{gamma}RI on THP1 cells. Three lines of evidence support this conclusion. Firstly, the binding of HRG to THP1 cells is inhibited by monomeric human or rabbit IgG which are known to interact only with Fc{gamma}RI. Secondly, mAb22 against Fc{gamma}RI (anti-CD64) partially inhibited (35%) the binding of HRG to THP1 cells (Fig. 1CGo). Thirdly, IgG does not interfere with the binding of HRG to the T cell lines Jurkat and MT4 which lack Fc{gamma}RI (not shown).

A major finding of the present study is that the pre-treatment of THP1 cells with HRG significantly inhibits the binding of monomeric human IgG to THP1 cells, with the binding being inhibited ~90% by 2 µM HRG. It should be noted that this observation is physiologically relevant as the plasma concentration of human HRG is ~2 µM. Interestingly, the pre-incubation of THP1 cells with HRG also inhibited the binding of both monomeric human IgG1 and IgG3 to these cells (not shown), but the presence of Zn2+ did not alter the effect of HRG on the interaction of IgG with THP1 cells (Fig. 2BGo, squares). Since monomeric IgG binds to Fc{gamma}RI on THP1 cells, these findings suggest that the pre-treatment of the THP1 cells with HRG can mask the IgG-binding site on the Fc{gamma}RI. Interestingly, the presence of IC in the reaction vessel did not change the ability of HRG to inhibit the subsequent binding of monomeric IgG to THP1 cells. However, the fact that pre-treatment of the cells with HRG did not affect the binding of IC to these cells may be explained by the ability of the IC to bind to low-affinity FcR (Fc{gamma}RII, etc.) on THP1 cells which may not be blocked by HRG. Another possibility is the established ability of IC to bind to cells multivalently and hence with higher avidity, a process which may displace HRG from Fc{gamma}RI on the cell surface.

Despite the fact that pre-incubation of THP1 cells with HRG inhibited subsequent binding of monomeric IgG, the binding of IgG to THP1 cells was potentiated when IgG was pre-incubated with HRG (Fig. 2BGo, triangles). Similarly, the pre-incubation of HRG with monomeric human IgG1 and IgG3 also resulted in an enhanced binding of these IgG subclasses to THP1 cells (not shown). The potentiating effect of HRG under these conditions is likely to be due to the binding of the IgG–HRG complexes to heparan sulfates on the cell surface via heparan sulfate binding sites on HRG (28,39). The data presented also show that the molar ratio of HRG:IgG is an important factor in determining whether HRG potentiates (HRG:IgG molar ratio ~ 1–10:1) or inhibits (HRG:IgG molar ratio >= 50:1) the binding of free IgG to Fc{gamma}RI. Thus, it would be expected that under physiological conditions plasma HRG (2 µM) would not enhance the binding of monomeric plasma IgG (~60 µM) to monocytes since the molar ratio of HRG:IgG is ~1:30.

Another major finding from the present work is that HRG potentiates the binding of IgG-containing IC to THP1 cells in the presence of physiological concentrations of Zn2+. IC containing FITC–IgG and OVA were used to study the effect of HRG on the interaction of IC with THP1 cells. Turbidity assays and precipitin curve analysis of insoluble IC formed using FITC–IgG and OVA indicated that insoluble IC formation in this system was indistinguishable from that which occurred when using unlabeled IgG and OVA (not shown). Also, the binding of IC to THP1 cells showed a similar pattern to the precipitin curve analysis of FITC–IgG-OVA IC (see Fig 3AGo, cf. circles with squares). These findings indicate that the binding of IC to Fc{gamma}R is dependent on the antigen:antibody ratio of the IC mixture and that maximum binding of IC occurs at or near the equivalence antigen:antibody ratio. This result is consistent with the observation that IC formed at an equivalence antigen:antibody ratio are more effective in stimulating mouse peritoneal macrophages (40).

IC containing FITC–IgG, OVA and HRG were found to bind to THP1 cells at higher levels than IC containing only FITC–IgG and OVA when 20 µM Zn2+ was present. The results show, therefore, that HRG promotes the binding of IC to THP1 cells in a Zn2+-dependent fashion. There are two possible explanation for this phenomenon. First, our previous studies showed that Zn2+ substantially enhances the binding of HRG to IgG (29). Second, Zn2+ enhances the binding of HRG to heparan sulfates structures on cell surfaces (28). HRG appears to maximally potentiate the binding of IC to monocytes when the IC are formed with a HRG:IgG molar ratio of ~1. However, at higher HRG:IgG molar ratios little or no enhancement of the binding of IC occurs and, in fact, when the HRG:IgG molar ratio was >=4 inhibition of IC binding was observed, presumably due to excess HRG masking FcR on monocytes.

To date the uptake of IC by the RES has been proposed to involve two mechanisms, i.e. the binding of IC to FcR and the binding of IC complexed with C3b to CR. The production of C3b or C3d requires activation of the complement cascade which results in the release of other factors (e.g. anaphylatoxins, membrane attack complex) not yet shown to be involved in the clearance of IC (3,10). The present study provides evidence for the existence of a third mechanism for enhancing the uptake of IC by monocytes, i.e. by incorporation of HRG in IC. Our results indicate that HRG can promote the binding of IC to FcR probably via binding to the glycosaminoglycan, heparan sulfate and possibly to other HRG receptors on the cell surface. The binding or incorporation of HRG into IC also may increase the avidity of these IC for the cell surface due to the formation of multivalent interactions between IC and heparan sulfate receptors for HRG.

The finding that HRG inhibits the binding of monomeric IgG to monocytes by masking Fc{gamma}RI is an intriguing observation. It seems likely that the region of HRG which binds IgG also binds Fc{gamma}RI since the extracellular portion of Fc{gamma}RI contains three Ig-like domains (37). Whether HRG can interact with FcR other than Fc{gamma}RI and regulate their function is currently under investigation. However, the normal physiological concentrations of HRG and IgG in plasma are such that most HRG would be associated with soluble IgG and unavailable to interact with Fc{gamma}RI.

It has been shown that the deposition of circulating IC in various organs (e.g. kidney, joint and the blood vessel wall) may lead to the development of pathological conditions such as glomerulonephritis, arthritis and vasculitis (41,42). It also has been shown that tissue IC deposits are in equilibrium with circulating IC (2,41) and that a reduction of IC in the circulation is necessary for therapy in the majority of IC-mediated diseases (43). Attenuation of the activation of complement also was proposed to be a clinical strategy to treat these pathological conditions (4447). Since HRG is a plasma protein, it is possible that in the treatment of IC-mediated diseases HRG may be used as a therapeutic agent for the prevention of the pathogenic effects of IC in terms of keeping IC in a soluble form and potentiating their uptake by the RES.


    Acknowledgments
 
The authors are grateful to Ms Anna Bezos and Mr Geoffrey Osborne for their technical assistance. This work was supported in part by a project grant (971019) to J. G. A. from NH & MRC of Australia.


    Abbreviations
 
bHRGbiotinylated HRG
bIgGbiotinylated IgG
CR1complement receptor 1
FIUfluorescence intensity unit
HRGhistidine-rich glycoprotein
ICimmune complexes
OVAovalbumin
RESreticuloendothelial system
STPstreptavidin

    Notes
 
Transmitting editor: A. Kelso

Received 12 January 1999, accepted 27 April 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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